Expression patterns of the aquaporin gene family during renal development: influence of genetic variability.

PubMed

Parreira, Kleber S; Debaix, Huguette; Cnops, Yvette; Geffers, Lars; Devuyst, Olivier

2009-08-01

High-throughput analyses have shown that aquaporins (AQPs) belong to a cluster of genes that are differentially expressed during kidney organogenesis. However, the spatiotemporal expression patterns of the AQP gene family during tubular maturation and the potential influence of genetic variation on these patterns and on water handling remain unknown. We investigated the expression patterns of all AQP isoforms in fetal (E13.5 to E18.5), postnatal (P1 to P28), and adult (9 weeks) kidneys of inbred (C57BL/6J) and outbred (CD-1) mice. Using quantitative polymerase chain reaction (PCR), we evidenced two mRNA patterns during tubular maturation in C57 mice. The AQPs 1-7-11 showed an early (from E14.5) and progressive increase to adult levels, similar to the mRNA pattern observed for proximal tubule markers (Megalin, NaPi-IIa, OAT1) and reflecting the continuous increase in renal cortical structures during development. By contrast, AQPs 2-3-4 showed a later (E15.5) and more abrupt increase, with transient postnatal overexpression. Most AQP genes were expressed earlier and/or stronger in maturing CD-1 kidneys. Furthermore, adult CD-1 kidneys expressed more AQP2 in the collecting ducts, which was reflected by a significant delay in excreting a water load. The expression patterns of proximal vs. distal AQPs and the earlier expression in the CD-1 strain were confirmed by immunoblotting and immunostaining. These data (1) substantiate the clustering of important genes during tubular maturation and (2) demonstrate that genetic variability influences the regulation of the AQP gene family during tubular maturation and water handling by the mature kidney.

Increased leaf mesophyll porosity following transient retinoblastoma-related protein silencing is revealed by microcomputed tomography imaging and leads to a system-level physiological response to the altered cell division pattern

PubMed Central

Dorca-Fornell, Carmen; Pajor, Radoslaw; Lehmeier, Christoph; Pérez-Bueno, Marísa; Bauch, Marion; Sloan, Jen; Osborne, Colin; Rolfe, Stephen; Sturrock, Craig; Mooney, Sacha; Fleming, Andrew

2013-01-01

The causal relationship between cell division and growth in plants is complex. Although altered expression of cell-cycle genes frequently leads to altered organ growth, there are many examples where manipulation of the division machinery leads to a limited outcome at the level of organ form, despite changes in constituent cell size. One possibility, which has been under-explored, is that altered division patterns resulting from manipulation of cell-cycle gene expression alter the physiology of the organ, and that this has an effect on growth. We performed a series of experiments on retinoblastoma-related protein (RBR), a well characterized regulator of the cell cycle, to investigate the outcome of altered cell division on leaf physiology. Our approach involved combination of high-resolution microCT imaging and physiological analysis with a transient gene induction system, providing a powerful approach for the study of developmental physiology. Our investigation identifies a new role for RBR in mesophyll differentiation that affects tissue porosity and the distribution of air space within the leaf. The data demonstrate the importance of RBR in early leaf development and the extent to which physiology adapts to modified cellular architecture resulting from altered cell-cycle gene expression. PMID:24118480

Transient expression of collagen type II at epitheliomesenchymal interfaces during morphogenesis of the cartilaginous neurocranium.

PubMed

Thorogood, P; Bee, J; von der Mark, K

1986-08-01

In the avian embryo a matrix-mediated tissue interaction between retinal pigmented epithelium and neural crest-derived periocular mesenchyme leads to the differentiation of (scleral) cartilage. The composition of the extracellular matrix at the interface between these two tissues has been examined immunohistochemically, both during and after the interaction has taken place. Of the matrix components studied (fibronectin, laminin, and collagen types I, II, IV, and V) only collagen type II displayed a dramatic change in distribution between the two stages. During the interaction, at stage 15, type II was present in the extracellular compartment basal to the epithelium. After completion of the interaction, collagen type II was no longer detectable at the interface even though it was readily detectable in the vitreous humor, cornea, and perinotochordal sheath, and subsequently will be expressed by the chondrogenic tissue itself as overt differentiation commences. These results suggest that collagen type II might be causally involved in this particular epitheliomesenchymal interaction. Examination of the spatial and temporal patterns of collagen type II expression elsewhere in the developing craniofacial complex revealed a hitherto unreported pattern of distribution. In addition to its predictable locations (i.e., cornea, vitreous, and perinotochordal sheath) it was found to be present at certain other sites, for example, at the basal surfaces of some neuroepithelia. These additional locations are all known to be sites of chondrogenesis-promoting tissue interactions which result in the formation of the elements of the cartilaginous neurocranium (e.g., otic vesicle). Furthermore this spatial distribution exhibits a changing temporal pattern in that it is detectable at the time that the interactions are known to be taking place, but subsequently is no longer detectable by the immunohistochemical means employed. This definable pattern of transient collagen type II expression, occurring at very early stages of craniofacial development, is interpreted as reflecting one level of morphogenetic specification of chondrocranial/skull form in the developing vertebrate head.

Rapid and systemic accumulation of chloroplast mRNA-binding protein transcripts after flame stimulus in tomato

NASA Technical Reports Server (NTRS)

Vian, A.; Henry-Vian, C.; Davies, E.

1999-01-01

It has been shown that tomato (Lycopersicon esculentum) plants respond to flame wounding and electrical stimulation by a rapid (15 min) and systemic up-regulation of proteinase inhibitor (pin) genes. To find other genes having a similar expression pattern, we used subtractive cDNA screening between flamed and control plants to select clones up-regulated by flame wounding. We report the characterization of one of them, a chloroplast mRNA-binding protein encoded by a single gene and expressed preferentially in the leaves. Systemic gene expression in response to flaming in the youngest terminal leaf exhibited three distinct phases: a rapid and transient increase (5-15 min) in transcript accumulation, a decline to basal levels (15-45 min), and then a second, more prolonged increase (60-90 min). In contrast, after a mechanical wound the rapid, transient increase (5 min) was followed by a rapid decline to basal levels but no later, prolonged accumulation. In the petiole, the initial flame-wound-evoked transient increase (15 min) was followed by a continuous decline for 3 h. The nature of the wound signal(s) causing such rapid changes in transcript abundance is discussed in relation to electrical signaling, which has recently been implicated in plant responses to wounding.

Timing Is Everything: Highly Specific and Transient Expression of a MAP Kinase Determines Auxin-Induced Leaf Venation Patterns in Arabidopsis

PubMed Central

Stanko, Vera; Giuliani, Concetta; Retzer, Katarzyna; Djamei, Armin; Wahl, Vanessa; Wurzinger, Bernhard; Wilson, Cathal; Heberle-Bors, Erwin; Teige, Markus; Kragler, Friedrich

2014-01-01

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules present in all eukaryotes. In plants, MAPK cascades were shown to regulate cell division, developmental processes, stress responses, and hormone pathways. The subgroup A of Arabidopsis MAPKs consists of AtMPK3, AtMPK6, and AtMPK10. AtMPK3 and AtMPK6 are activated by their upstream MAP kinase kinases (MKKs) AtMKK4 and AtMKK5 in response to biotic and abiotic stress. In addition, they were identified as key regulators of stomatal development and patterning. AtMPK10 has long been considered as a pseudo-gene, derived from a gene duplication of AtMPK6. Here we show that AtMPK10 is expressed highly but very transiently in seedlings and at sites of local auxin maxima leaves. MPK10 encodes a functional kinase and interacts with the upstream MAP kinase kinase (MAPKK) AtMKK2. mpk10 mutants are delayed in flowering in long-day conditions and in continuous light. Moreover, cotyledons of mpk10 and mkk2 mutants have reduced vein complexity, which can be reversed by inhibiting polar auxin transport (PAT). Auxin does not affect AtMPK10 expression while treatment with the PAT inhibitor HFCA extends the expression in leaves and reverses the mpk10 mutant phenotype. These results suggest that the AtMKK2–AtMPK10 MAPK module regulates venation complexity by altering PAT efficiency. PMID:25064848

Clustering of Ca2+ transients in interstitial cells of Cajal defines slow wave duration

PubMed Central

Drumm, Bernard T.; Hennig, Grant W.; Battersby, Matthew J.; Sung, Tae Sik

2017-01-01

Interstitial cells of Cajal (ICC) in the myenteric plexus region (ICC-MY) of the small intestine are pacemakers that generate rhythmic depolarizations known as slow waves. Slow waves depend on activation of Ca2+-activated Cl− channels (ANO1) in ICC, propagate actively within networks of ICC-MY, and conduct to smooth muscle cells where they generate action potentials and phasic contractions. Thus, mechanisms of Ca2+ regulation in ICC are fundamental to the motor patterns of the bowel. Here, we characterize the nature of Ca2+ transients in ICC-MY within intact muscles, using mice expressing a genetically encoded Ca2+ sensor, GCaMP3, in ICC. Ca2+ transients in ICC-MY display a complex firing pattern caused by localized Ca2+ release events arising from multiple sites in cell somata and processes. Ca2+ transients are clustered within the time course of slow waves but fire asynchronously during these clusters. The durations of Ca2+ transient clusters (CTCs) correspond to slow wave durations (plateau phase). Simultaneous imaging and intracellular electrical recordings revealed that the upstroke depolarization of slow waves precedes clusters of Ca2+ transients. Summation of CTCs results in relatively uniform Ca2+ responses from one slow wave to another. These Ca2+ transients are caused by Ca2+ release from intracellular stores and depend on ryanodine receptors as well as amplification from IP3 receptors. Reduced extracellular Ca2+ concentrations and T-type Ca2+ channel blockers decreased the number of firing sites and firing probability of Ca2+ transients. In summary, the fundamental electrical events of small intestinal muscles generated by ICC-MY depend on asynchronous firing of Ca2+ transients from multiple intracellular release sites. These events are organized into clusters by Ca2+ influx through T-type Ca2+ channels to sustain activation of ANO1 channels and generate the plateau phase of slow waves. PMID:28592421

Differential skeletal muscle proteome of high- and low-active mice

PubMed Central

Dangott, Lawrence J.; Schmitt, Emily E.; Vellers, Heather L.; Lightfoot, J. Timothy

2014-01-01

Physical inactivity contributes to cardiovascular disease, type II diabetes, obesity, and some types of cancer. While the literature is clear that there is genetic regulation of physical activity with existing gene knockout data suggesting that skeletal muscle mechanisms contribute to the regulation of activity, actual differences in end-protein expression between high- and low-active mice have not been investigated. This study used two-dimensional differential gel electrophoresis coupled with mass spectrometry to evaluate the proteomic differences between high-active (C57L/J) and low-active (C3H/HeJ) mice in the soleus and extensor digitorum longus (EDL). Furthermore, vivo-morpholinos were used to transiently knockdown candidate proteins to confirm their involvement in physical activity regulation. Proteins with higher expression patterns generally fell into the calcium-regulating and Krebs (TCA) cycle pathways in the high-active mice (e.g., annexin A6, P = 0.0031; calsequestrin 1; P = 0.000025), while the overexpressed proteins in the low-active mice generally fell into cytoskeletal structure- and electron transport chain-related pathways (e.g., ATPase, P = 0.031; NADH dehydrogenase, P = 0.027). Transient knockdown of annexin A6 and calsequestrin 1 protein of high-active mice with vivo-morpholinos resulted in decreased physical activity levels (P = 0.001). These data suggest that high- and low-active mice have unique protein expression patterns and that each pattern contributes to the peripheral capability to be either high- or low-active, suggesting that different specific mechanisms regulate activity leading to the high- or low-activity status of the animal. PMID:24505100

Transitional gene expression profiling in ovarian follicle during ovulation in normal-cycle rats.

PubMed

Tsubota, Kenjiro; Kanki, Masayuki; Noto, Takahisa; Shiraki, Katsuhisa; Takeuchi, Ayano; Nakatsuji, Shunji; Seki, Jiro; Oishi, Yuji; Matsumoto, Masahiro; Nakayama, Hiroyuki

2011-06-01

Evaluation of ovarian toxicity requires an understanding of the physiological changes related to the estrous cycle in the ovary. The authors investigated the transitional gene expression profile of ovulatory follicles in rats that show normal estrous cyclicity. Ovaries were collected at 10:00 and 22:00 on the proestrus day and at 10:00 on the estrus day. Ovarian follicles or early corpora lutea were isolated using laser microdissection, and extracted total RNA was analyzed using microarray technology. Clustering analysis revealed four different expression patterns: transient up- or down-regulation only at 22:00 on the proestrus day (pattern 1), up- or down-regulation only at 10:00 on the estrus day (pattern 2), continuous increase at 22:00 on the proestrus day and at 10:00 on the estrus day (pattern 3), and up- or down-regulation at 22:00 on the proestrus day and level maintenance at 10:00 on the estrus day (pattern 4). In addition, these probe sets were functionally categorized in each pattern using the Ingenuity Pathways Analysis database. These data will aid in understanding the physiology of ovulation and may be useful in assessing ovarian toxicity and its mechanism, such as in investigations of chemical-induced ovulatory impairment.

An amphioxus Msx gene expressed predominantly in the dorsal neural tube.

PubMed

Sharman, A C; Shimeld, S M; Holland, P W

1999-04-01

Genomic and cDNA clones of an Msx class homeobox gene were isolated from amphioxus (Branchiostoma floridae). The gene, AmphiMsx, is expressed in the neural plate from late gastrulation; in later embryos it is expressed in dorsal cells of the neural tube, excluding anterior and posterior regions, in an irregular reiterated pattern. There is transient expression in dorsal cells within somites, reminiscent of migrating neural crest cells of vertebrates. In larvae, mRNA is detected in two patches of anterior ectoderm proposed to be placodes. Evolutionary analyses show there is little phylogenetic information in Msx protein sequences; however, it is likely that duplication of Msx genes occurred in the vertebrate lineage.

Effect of transient receptor potential vanilloid 6 gene silencing on the expression of calcium transport genes in chicken osteoblasts.

PubMed

Zhang, Jie; Deng, Yifeng; Ma, Huijie; Hou, Jiafa; Zhou, ZhenLei

2015-03-01

Ca2+ plays a major role in the regulation of signal transduction. Transient receptor potential vanilloid 6 is a Ca2+-selective channel that serves as an important rate-limiting step in the facilitation of Ca2+ entry into cells, but little is known about the regulation of transient receptor potential vanilloid 6 in chickens. In this study, we evaluated the effects of transient receptor potential vanilloid 6 gene interference on the expression of calbindin-D28K, Na+/Ca2+ exchangers, and plasma membrane Ca2+ ATPase 1b to investigate the mechanism underlying the regulation of transient receptor potential vanilloid 6. Three hairpin siRNA expression vectors targeting transient receptor potential vanilloid 6 (pSIREN- transient receptor potential vanilloid 6) and a negative control (pSIREN-control) were constructed and transfected into chicken osteoblasts. The mRNA and protein expression levels were evaluated by quantitative reverse transcription polymerase chain reaction and Western blot, respectively. The mRNA expression levels of transient receptor potential vanilloid 6 and calbindin-D28K were reduced by 45.7% (P<0.01) and 27.9% (P<0.01), respectively, 48 h after transfection with one of the three constructs (pSIREN- transient receptor potential vanilloid 6-3) compared with the level obtained in the untreated group. There was no significant difference in the mRNA expression levels of Na+/Ca2+ exchangers and plasma membrane Ca2+ ATPase 1b. The protein expression levels of transient receptor potential vanilloid 6 and calbindin-D28K were reduced by 40.2% (P<0.01) and 29.8% (P<0.01), respectively, 48 h after transfection with pSIREN-transient receptor potential vanilloid 6-3 compared with the level obtained in the untreated group. In conclusion, the vector-based transient receptor potential vanilloid 6-shRNA can efficiently suppress the mRNA and protein expression of transient receptor potential vanilloid 6 in chicken osteoblasts, and transient receptor potential vanilloid 6 regulates the expression of calbindin-D28K during Ca2+ transport. © 2015 Poultry Science Association Inc.

Dendrimer-driven neurotrophin expression differs in temporal patterns between rodent and human stem cells.

PubMed

Shakhbazau, Antos; Shcharbin, Dzmitry; Seviaryn, Ihar; Goncharova, Natalya; Kosmacheva, Svetlana; Potapnev, Mihail; Bryszewska, Maria; Kumar, Ranjan; Biernaskie, Jeffrey; Midha, Rajiv

2012-05-07

This study reports the use of a nonviral expression system based on polyamidoamine dendrimers for time-restricted neurotrophin overproduction in mesenchymal stem cells and skin precursor-derived Schwann cells. The dendrimers were used to deliver plasmids for brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) expression in both rodent and human stem cells, and the timelines of expression were studied. We have found that, despite the fact that transfection efficiencies and protein expression levels were comparable, dendrimer-driven expression in human mesenchymal stem cells was characterized by a more rapid decline compared to rodent cells. Transient expression systems can be beneficial for some neurotrophins, which were earlier reported to cause unwanted side effects in virus-based long-term expression models. Nonviral neurotrophin expression is a biologically safe and accessible alternative to increase the therapeutic potential of autologous adult stem cells and stem cell-derived functional differentiated cells.

Dynamic Maternal Gradients Control Timing and Shift-Rates for Drosophila Gap Gene Expression

PubMed Central

Verd, Berta; Crombach, Anton

2017-01-01

Pattern formation during development is a highly dynamic process. In spite of this, few experimental and modelling approaches take into account the explicit time-dependence of the rules governing regulatory systems. We address this problem by studying dynamic morphogen interpretation by the gap gene network in Drosophila melanogaster. Gap genes are involved in segment determination during early embryogenesis. They are activated by maternal morphogen gradients encoded by bicoid (bcd) and caudal (cad). These gradients decay at the same time-scale as the establishment of the antero-posterior gap gene pattern. We use a reverse-engineering approach, based on data-driven regulatory models called gene circuits, to isolate and characterise the explicitly time-dependent effects of changing morphogen concentrations on gap gene regulation. To achieve this, we simulate the system in the presence and absence of dynamic gradient decay. Comparison between these simulations reveals that maternal morphogen decay controls the timing and limits the rate of gap gene expression. In the anterior of the embyro, it affects peak expression and leads to the establishment of smooth spatial boundaries between gap domains. In the posterior of the embryo, it causes a progressive slow-down in the rate of gap domain shifts, which is necessary to correctly position domain boundaries and to stabilise the spatial gap gene expression pattern. We use a newly developed method for the analysis of transient dynamics in non-autonomous (time-variable) systems to understand the regulatory causes of these effects. By providing a rigorous mechanistic explanation for the role of maternal gradient decay in gap gene regulation, our study demonstrates that such analyses are feasible and reveal important aspects of dynamic gene regulation which would have been missed by a traditional steady-state approach. More generally, it highlights the importance of transient dynamics for understanding complex regulatory processes in development. PMID:28158178

Dynamic Maternal Gradients Control Timing and Shift-Rates for Drosophila Gap Gene Expression.

PubMed

Verd, Berta; Crombach, Anton; Jaeger, Johannes

2017-02-01

Pattern formation during development is a highly dynamic process. In spite of this, few experimental and modelling approaches take into account the explicit time-dependence of the rules governing regulatory systems. We address this problem by studying dynamic morphogen interpretation by the gap gene network in Drosophila melanogaster. Gap genes are involved in segment determination during early embryogenesis. They are activated by maternal morphogen gradients encoded by bicoid (bcd) and caudal (cad). These gradients decay at the same time-scale as the establishment of the antero-posterior gap gene pattern. We use a reverse-engineering approach, based on data-driven regulatory models called gene circuits, to isolate and characterise the explicitly time-dependent effects of changing morphogen concentrations on gap gene regulation. To achieve this, we simulate the system in the presence and absence of dynamic gradient decay. Comparison between these simulations reveals that maternal morphogen decay controls the timing and limits the rate of gap gene expression. In the anterior of the embyro, it affects peak expression and leads to the establishment of smooth spatial boundaries between gap domains. In the posterior of the embryo, it causes a progressive slow-down in the rate of gap domain shifts, which is necessary to correctly position domain boundaries and to stabilise the spatial gap gene expression pattern. We use a newly developed method for the analysis of transient dynamics in non-autonomous (time-variable) systems to understand the regulatory causes of these effects. By providing a rigorous mechanistic explanation for the role of maternal gradient decay in gap gene regulation, our study demonstrates that such analyses are feasible and reveal important aspects of dynamic gene regulation which would have been missed by a traditional steady-state approach. More generally, it highlights the importance of transient dynamics for understanding complex regulatory processes in development.

Separable roles of UFO during floral development revealed by conditional restoration of gene function.

PubMed

Laufs, Patrick; Coen, Enrico; Kronenberger, Jocelyne; Traas, Jan; Doonan, John

2003-02-01

The UNUSUAL FLORAL ORGANS (UFO) gene is required for several aspects of floral development in Arabidopsis including specification of organ identity in the second and third whorls and the proper pattern of primordium initiation in the inner three whorls. UFO is expressed in a dynamic pattern during the early phases of flower development. Here we dissect the role of UFO by ubiquitously expressing it in ufo loss-of-function flowers at different developmental stages and for various durations using an ethanol-inducible expression system. The previously known functions of UFO could be separated and related to its expression at specific stages of development. We show that a 24- to 48-hour period of UFO expression from floral stage 2, before any floral organs are visible, is sufficient to restore normal petal and stamen development. The earliest requirement for UFO is during stage 2, when the endogenous UFO gene is transiently expressed in the centre of the wild-type flower and is required to specify the initiation patterns of petal, stamen and carpel primordia. Petal and stamen identity is determined during stages 2 or 3, when UFO is normally expressed in the presumptive second and third whorl. Although endogenous UFO expression is absent from the stamen whorl from stage 4 onwards, stamen identity can be restored by UFO activation up to stage 6. We also observed floral phenotypes not observed in loss-of-function or constitutive gain-of-function backgrounds, revealing additional roles of UFO in outgrowth of petal primordia.

Epidermal patterning genes are active during embryogenesis in Arabidopsis.

PubMed

Costa, Silvia; Dolan, Liam

2003-07-01

Epidermal cells in the root of Arabidopsis seedling differentiate either as hair or non-hair cells, while in the hypocotyl they become either stomatal or elongated cells. WEREWOLF (WER) and GLABRA2 (GL2) are positive regulators of non-hair and elongated cell development. CAPRICE (CPC) is a positive regulator of hair cell development in the root. We show that WER, GL2 and CPC are expressed and active during the stages of embryogenesis when the pattern of cells in the epidermis of the root-hypocotyl axis forms. GL2 is first expressed in the future epidermis in the heart stage embryo and its expression is progressively restricted to those cells that will acquire a non-hair identity in the transition between torpedo and mature stage. The expression of GL2 at the heart stage requires WER function. WER and CPC are transiently expressed throughout the root epidermal layer in the torpedo stage embryo when the cell-specific pattern of GL2 expression is being established in the epidermis. We also show that WER positively regulates CPC transcription and GL2 negatively regulates WER transcription in the mature embryo. We propose that the restriction of GL2 to the future non-hair cells in the root epidermis can be correlated with the activities of WER and CPC during torpedo stage. In the embryonic hypocotyl we show that WER controls GL2 expression. We also provide evidence indicating that CPC may also regulate GL2 expression in the hypocotyl.

Characterization and promoter activity of chromoplast specific carotenoid associated gene (CHRC) from Oncidium Gower Ramsey.

PubMed

Chiou, Chung-Yi; Wu, Keqiang; Yeh, Kai-Wun

2008-10-01

Tissue-specific promoters are required for plant molecular breeding to drive a target gene in the appropriate location in plants. A chromoplast-specific, carotenoid-associated gene (OgCHRC) and its promoter (Pchrc) were isolated from Oncidium orchid and characterized. Northern blot analysis revealed that OgCHRC is specifically expressed in flowers, not in roots and leaves. Transient expression assay of Pchrc by bombardment transformation confirmed its differential expression pattern in floral tissues of different horticulture plants and cell-type location in conical papillate cells of adaxial epidermis of flower. These results suggest that Pchrc could serve as a useful tool in ornamental plant biotechnology to modify flower color.

Transient expression of progesterone receptor and cathepsin-l in human granulosa cells during the periovulatory period.

PubMed

García, Víctor; Kohen, Paulina; Maldonado, Carola; Sierralta, Walter; Muñoz, Alex; Villarroel, Claudio; Strauss, Jerome F; Devoto, Luigi

2012-03-01

To study in vivo the progesterone receptor (PR) expression levels in human granulosa cells (GCs) during the periovulatory period and the affect of the protein kinase A (PKA) pathway on PR expression and cathepsin-L expression-activation. Experimental study. University research unit. Twenty-five women of reproductive age. Follicular fluid and GCs obtained from spontaneous cycles before and during the normal luteinizing hormone surge, and samples obtained 36 hours after human chorionic gonadotropin (hCG) administration in patients undergoing in vitro fertilization. To determine PR, cathepsin-L messenger RNA (mRNA) analysis via real-time polymerase chain reaction, and protein of PR, cathepsin-L, and PKA in human GCs. The Western blot analysis revealed that bands of PR (isoform A) were the most abundant and that mRNA (PR-A and PR-B) have a temporal pattern of expression throughout the periovulatory period. The protein levels of PR and cathepsin-L were up-regulated by hCG. The abundance of PR was diminished in the presence of PKA inhibitor, and cathepsin-L with PR receptor antagonist. The transient expression of PR in human GCs of the preovulatory follicle suggests that PR and its ligand play a role in the activation of cathepsin-L, which is presumably involved in the degradation of the follicular extracellular matrix during human ovulation. Copyright © 2012 American Society for Reproductive Medicine. All rights reserved.

Tomato Sl3-MMP, a member of the Matrix metalloproteinase family, is required for disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000.

PubMed

Li, Dayong; Zhang, Huijuan; Song, Qiuming; Wang, Lu; Liu, Shixia; Hong, Yongbo; Huang, Lei; Song, Fengming

2015-06-14

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. MMPs have been characterized in detail in mammals and shown to play key roles in many physiological and pathological processes. Although MMPs in some plant species have been identified, the function of MMPs in biotic stress responses remains elusive. A total of five MMP genes were identified in tomato genome. qRT-PCR analysis revealed that expression of Sl-MMP genes was induced with distinct patterns by infection of Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 and by treatment with defense-related hormones such as salicylic acid, jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Virus-induced gene silencing (VIGS)-based knockdown of individual Sl-MMPs and disease assays indicated that silencing of Sl3-MMP resulted in reduced resistance to B. cinerea and Pst DC3000, whereas silencing of other four Sl-MMPs did not affect the disease resistance against these two pathogens. The Sl3-MMP-silenced tomato plants responded with increased accumulation of reactive oxygen species and alerted expression of defense genes after infection of B. cinerea. Transient expression of Sl3-MMP in leaves of Nicotiana benthamiana led to an enhanced resistance to B. cinerea and upregulated expression of defense-related genes. Biochemical assays revealed that the recombinant mature Sl3-MMP protein had proteolytic activities in vitro with distinct preferences for specificity of cleavage sites. The Sl3-MMP protein was targeted onto the plasma membrane of plant cells when transiently expressed in onion epidermal cells. VIGS-based knockdown of Sl3-MMP expression in tomato and gain-of-function transient expression of Sl3-MMP in N. benthamiana demonstrate that Sl3-MMP functions as a positive regulator of defense response against B. cinerea and Pst DC3000.

CXCR4 expression is associated with time-course permanent and temporary myocardial infarction in rats.

PubMed

Kiani, Ali Asghar; Babaei, Fereshteh; Sedighi, Mehrnoosh; Soleimani, Azam; Ahmadi, Kolsum; Shahrokhi, Somayeh; Anbari, Khatereh; Nazari, Afshin

2017-06-01

Experimental myocardial infarction triggers secretion of Stromal cell-derived factor1 and lead to increase in the expression of its receptor "CXCR4" on the surface of various cells. The aim of this study was to evaluate the expression pattern of CXCR4 in peripheral blood cells following time-course permanent and temporary ischemia in rats. Fourteen male Wistar rats were divided into two groups of seven and were placed under permanent and transient ischemia. Peripheral blood mononuclear cells were isolated at different time points, RNAs extracted and applied to qRT-PCR analysis of the CXCR4 gene. Based on repeated measures analysis of variance, the differences in the expression levels of the gene in each of the groups were statistically significant over time (the effect of time) ( P <0.001). Additionally, the difference in gene expression between the two groups was statistically significant (the effect of group), such that at all times, the expression levels of the gene were significantly higher in the permanent ischemia than in the transient ischemia group ( P <0.001). Moreover, the interactive effect of time-group on gene expression was statistically significant ( P <0.001). CXCR4 is modulated in an induced ischemia context implying a possible association with myocardial infarction. Checking of CXCR4 expression in the ischemic changes shows that damage to the heart tissue trigger the secretion of inflammatory chemokine SDF, Followed by it CXCR4 expression in blood cells. These observations suggest that changes in the expression of CXCR4 may be considered a valuable marker for monitoring myocardial infarction.

Effect of leaf incubation temperature profiles on Agrobacterium tumefaciens-mediated transient expression.

PubMed

Jung, Sang-Kyu; McDonald, Karen A; Dandekar, Abhaya M

2015-01-01

Agrobacterium tumefaciens-mediated transient expression is known to be highly dependent on incubation temperature. Compared with early studies that were conducted at constant temperature, we examined the effect of variable leaf incubation temperature on transient expression. As a model system, synthetic endoglucanase (E1) and endoxylanase (Xyn10A) genes were transiently expressed in detached whole sunflower leaves via vacuum infiltration for biofuel applications. We found that the kinetics of transient expression strongly depended on timing of the temperature change as well as leaf incubation temperature. Surprisingly, we found that high incubation temperature (27-30 °C) which is suboptimal for T-DNA transfer, significantly enhanced transient expression if the high temperature was applied during the late phase (Day 3-6) of leaf incubation whereas incubation temperature in a range of 20-25 °C for an early phase (Day 0-2) resulted in higher production. On the basis of these results, we propose that transient expression is governed by both T-DNA transfer and protein synthesis in plant cells that have different temperature dependent kinetics. Because the phases were separated in time and had different optimal temperatures, we were then able to develop a novel two phase optimization strategy for leaf incubation temperature. Applying the time-varying temperature profile, we were able to increase the protein accumulation by fivefold compared with the control at a constant temperature of 20 °C. From our knowledge, this is the first report illustrating the effect of variable temperature profiling for improved transient expression. © 2015 American Institute of Chemical Engineers.

Detailed analysis of the δ-crystallin mRNA-expressing region in early development of the chick pituitary gland.

PubMed

Inoue, Makiko; Shiina, Tomoya; Aizawa, Sayaka; Sakata, Ichiro; Takagi, Hiroyasu; Sakai, Takafumi

2012-06-01

Although δ-crystallin (δ-crys), also known as lens protein, is transiently expressed in Rathke's pouch (RP) of the chick embryo, detailed temporal and spatial expression patterns have been obscure. In this study, to understand the relationship between the δ-crys mRNA-expressing region and RP formation, we examined the embryonic expression pattern of δ-crys mRNA in the primordium of the adenohypophysis. δ-crys mRNA expression was initially found at stage 15 anterior to the foregut and posterior to the invaginated oral ectoderm. After RP formation, the δ-crys mRNA was expressed in the post-ventral region of RP and the anterior region of RP. δ-crys mRNA expression was then restricted to the cephalic lobe of the pituitary gland. From stage 20, the δ-crys and alpha-glycoprotein subunit (αGSU) mRNA-expressing regions were almost completely overlapping. The αGSU mRNA-expressing region is thought to be the primordium of the pars tuberalis, and these regions were overlapped with the Lhx3 mRNA-expressing region. The intensity of δ-crys mRNA expression gradually decreased with development and completely disappeared by stage 34. These results suggest that the embryonic chick pituitary gland consists of two different regions labeled with δ-crys and Lhx3.

Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells

PubMed Central

Takayama, Naoya; Nishimura, Satoshi; Nakamura, Sou; Shimizu, Takafumi; Ohnishi, Ryoko; Endo, Hiroshi; Yamaguchi, Tomoyuki; Otsu, Makoto; Nishimura, Ken; Nakanishi, Mahito; Sawaguchi, Akira; Nagai, Ryozo; Takahashi, Kazutoshi; Yamanaka, Shinya; Nakauchi, Hiromitsu

2010-01-01

Human (h) induced pluripotent stem cells (iPSCs) are a potentially abundant source of blood cells, but how best to select iPSC clones suitable for this purpose from among the many clones that can be simultaneously established from an identical source is not clear. Using an in vitro culture system yielding a hematopoietic niche that concentrates hematopoietic progenitors, we show that the pattern of c-MYC reactivation after reprogramming influences platelet generation from hiPSCs. During differentiation, reduction of c-MYC expression after initial reactivation of c-MYC expression in selected hiPSC clones was associated with more efficient in vitro generation of CD41a+CD42b+ platelets. This effect was recapitulated in virus integration-free hiPSCs using a doxycycline-controlled c-MYC expression vector. In vivo imaging revealed that these CD42b+ platelets were present in thrombi after laser-induced vessel wall injury. In contrast, sustained and excessive c-MYC expression in megakaryocytes was accompanied by increased p14 (ARF) and p16 (INK4A) expression, decreased GATA1 expression, and impaired production of functional platelets. These findings suggest that the pattern of c-MYC expression, particularly its later decline, is key to producing functional platelets from selected iPSC clones. PMID:21098095

Analysis of Microbe-Associated Molecular Pattern-Responsive Synthetic Promoters with the Parsley Protoplast System.

PubMed

Kanofsky, Konstantin; Lehmeyer, Mona; Schulze, Jutta; Hehl, Reinhard

2016-01-01

Plants recognize pathogens by microbe-associated molecular patterns (MAMPs) and subsequently induce an immune response. The regulation of gene expression during the immune response depends largely on cis-sequences conserved in promoters of MAMP-responsive genes. These cis-sequences can be analyzed by constructing synthetic promoters linked to a reporter gene and by testing these constructs in transient expression systems. Here, the use of the parsley (Petroselinum crispum) protoplast system for analyzing MAMP-responsive synthetic promoters is described. The synthetic promoter consists of four copies of a potential MAMP-responsive cis-sequence cloned upstream of a minimal promoter and the uidA reporter gene. The reporter plasmid contains a second reporter gene, which is constitutively expressed and hence eliminates the requirement of a second plasmid used as a transformation control. The reporter plasmid is transformed into parsley protoplasts that are elicited by the MAMP Pep25. The MAMP responsiveness is validated by comparing the reporter gene activity from MAMP-treated and untreated cells and by normalizing reporter gene activity using the constitutively expressed reporter gene.

Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells.

PubMed

Hacker, David L; Bertschinger, Martin; Baldi, Lucia; Wurm, Florian M

2004-10-27

Human embryonic kidney 293 (HEK293) cells, a widely used host for large-scale transient expression of recombinant proteins, are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 EBNA (HEK293E) cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNA interference (RNAi). The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1alpha (EF-1alpha) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to twofold. E1A mRNA depletion also resulted in a twofold increase in transient expression of a recombinant IgG in both small- and large-scale suspension cultures when the IgG light and heavy chain genes were controlled by the EF-1alpha promoter. Finally, transient IgG expression was enhanced 2.5-fold when the anti-E1A shRNA was expressed from the same vector as the IgG light chain gene. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells, and they established that RNAi can be used to enhance recombinant protein expression in mammalian cells.

Pre-gastrula expression of zebrafish extraembryonic genes

PubMed Central

2010-01-01

Background Many species form extraembryonic tissues during embryogenesis, such as the placenta of humans and other viviparous mammals. Extraembryonic tissues have various roles in protecting, nourishing and patterning embryos. Prior to gastrulation in zebrafish, the yolk syncytial layer - an extraembryonic nuclear syncytium - produces signals that induce mesoderm and endoderm formation. Mesoderm and endoderm precursor cells are situated in the embryonic margin, an external ring of cells along the embryo-yolk interface. The yolk syncytial layer initially forms below the margin, in a domain called the external yolk syncytial layer (E-YSL). Results We hypothesize that key components of the yolk syncytial layer's mesoderm and endoderm inducing activity are expressed as mRNAs in the E-YSL. To identify genes expressed in the E-YSL, we used microarrays to compare the transcription profiles of intact pre-gastrula embryos with pre-gastrula embryonic cells that we had separated from the yolk and yolk syncytial layer. This identified a cohort of genes with enriched expression in intact embryos. Here we describe our whole mount in situ hybridization analysis of sixty-eight of them. This includes ten genes with E-YSL expression (camsap1l1, gata3, znf503, hnf1ba, slc26a1, slc40a1, gata6, gpr137bb, otop1 and cebpa), four genes with expression in the enveloping layer (EVL), a superficial epithelium that protects the embryo (zgc:136817, zgc:152778, slc14a2 and elovl6l), three EVL genes whose expression is transiently confined to the animal pole (elovl6l, zgc:136359 and clica), and six genes with transient maternal expression (mtf1, wu:fj59f04, mospd2, rftn2, arrdc1a and pho). We also assessed the requirement of Nodal signaling for the expression of selected genes in the E-YSL, EVL and margin. Margin expression was Nodal dependent for all genes we tested, including the concentrated margin expression of an EVL gene: zgc:110712. All other instances of EVL and E-YSL expression that we tested were Nodal independent. Conclusion We have devised an effective strategy for enriching and identifying genes expressed in the E-YSL of pre-gastrula embryos. To our surprise, maternal genes and genes expressed in the EVL were also enriched by this strategy. A number of these genes are promising candidates for future functional studies on early embryonic patterning. PMID:20423468

Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression.

PubMed

Sindarovska, Y R; Gerasymenko, I M; Sheludko, Y V; Olevinskaya, Z M; Spivak, N Y; Kuchuk, N V

2010-01-01

Human interferon alpha2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 x 10(3) IU/g fresh weight (FW) with an average of 2.1 +/- 0.8 x 10(3) IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN alpha2b.

Increases of Catalase and Glutathione Peroxidase Expressions by Lacosamide Pretreatment Contributes to Neuroprotection Against Experimentally Induced Transient Cerebral Ischemia.

PubMed

Choi, Hyun Young; Park, Joon Ha; Chen, Bai Hui; Shin, Bich Na; Lee, Yun Lyul; Kim, In Hye; Cho, Jeong-Hwi; Lee, Tae-Kyeong; Lee, Jae-Chul; Won, Moo-Ho; Ahn, Ji Hyeon; Tae, Hyun-Jin; Yan, Bing Chun; Hwang, In Koo; Cho, Jun Hwi; Kim, Young-Myeong; Kim, Sung Koo

2016-09-01

Lacosamide is a new antiepileptic drug which is widely used to treat partial-onset seizures. In this study, we examined the neuroprotective effect of lacosamide against transient ischemic damage and expressions of antioxidant enzymes such as Zn-superoxide dismutase (SOD1), Mn-superoxide dismutase (SOD2), catalase (CAT) and glutathione peroxidase (GPX) in the hippocampal cornu ammonis 1 (CA1) region following 5 min of transient global cerebral ischemia in gerbils. We found that pre-treatment with 25 mg/kg lacosamide protected CA1 pyramidal neurons from transient global cerebral ischemic insult using hematoxylin-eosin staining and neuronal nuclear antigen immunohistochemistry. Transient ischemia dramatically changed expressions of SOD1, SOD2 and GPX, not CAT, in the CA1 pyramidal neurons. Lacosamide pre-treatment increased expressions of CAT and GPX, not SOD1 and 2, in the CA1 pyramidal neurons compared with controls, and their expressions induced by lacosamide pre-treatment were maintained after transient cerebral ischemia. In brief, pre-treatment with lacosamide protected hippocampal CA1 pyramidal neurons from ischemic damage induced by transient global cerebral ischemia, and the lacosamide-mediated neuroprotection may be closely related to increases of CAT and GPX expressions by lacosamide pre-treatment.

Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

PubMed Central

Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

2016-01-01

This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

CitAP2.10 activation of the terpene synthase CsTPS1 is associated with the synthesis of (+)-valencene in ‘Newhall’ orange

PubMed Central

Shen, Shu-ling; Yin, Xue-ren; Zhang, Bo; Xie, Xiu-lan; Jiang, Qian; Grierson, Donald; Chen, Kun-song

2016-01-01

Aroma is a vital characteristic that determines the quality and commercial value of citrus fruits, and characteristic volatiles have been analyzed in different citrus species. In sweet orange, Citrus sinensis, the sesquiterpene (+)-valencene is a key volatile compound in the fruit peel. Valencene synthesis is catalyzed by the terpene synthase CsTPS1, but the transcriptional mechanisms controlling its gene expression are unknown. Here, the AP2/ERF (APETALA2/ethylene response factor) transcription factor, CitAP2.10, is characterized as a regulator of (+)-valencene synthesis. The expression pattern of CitAP2.10 was positively correlated with (+)-valencene content and CsTPS1 expression. Dual-luciferase assays indicated that CitAP2.10 could trans-activate the CsTPS1 promoter. Ethylene enhanced expression of CitAP2.10 and this effect was abolished by the ethylene antagonist 1-methylcyclopropene. The role and function of CitAP2.10 in (+)-valencene biosynthesis were confirmed using the Arabidopsis homolog (AtWRI1), which also transiently activated the CsTPS1 promoter. Furthermore, transient over-expression of CitAP2.10 triggered (+)-valencene biosynthesis in sweet orange fruit. These results indicate that CitAP2.10 regulates (+)-valencene synthesis via induction of CsTPS1 mRNA accumulation. PMID:27194737

Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme.

PubMed

Gallage, Nethaji J; Hansen, Esben H; Kannangara, Rubini; Olsen, Carl Erik; Motawia, Mohammed Saddik; Jørgensen, Kirsten; Holme, Inger; Hebelstrup, Kim; Grisoni, Michel; Møller, Birger Lindberg

2014-06-19

Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco.

Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme

PubMed Central

Gallage, Nethaji J.; Hansen, Esben H.; Kannangara, Rubini; Olsen, Carl Erik; Motawia, Mohammed Saddik; Jørgensen, Kirsten; Holme, Inger; Hebelstrup, Kim; Grisoni, Michel; Møller, Birger Lindberg

2014-01-01

Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco. PMID:24941968

Methylation-Dependent Activation of CDX1 through NF-κB

PubMed Central

Rau, Tilman T.; Rogler, Anja; Frischauf, Myrjam; Jung, Andreas; Konturek, Peter C.; Dimmler, Arno; Faller, Gerhard; Sehnert, Bettina; El-Rifai, Wael; Hartmann, Arndt; Voll, Reinhard E.; Schneider-Stock, Regine

2013-01-01

The caudal homeobox factor 1 (CDX1) is an essential transcription factor for intestinal differentiation. Its aberrant expression in intestinal metaplasia of the upper gastrointestinal tract is a hallmark within the gastritis-metaplasia-carcinoma sequence. CDX1 expression is influenced by certain pathways, such as Wnt, Ras, or NF-κB signaling; however, these pathways alone cannot explain the transient expression of CDX1 in intestinal metaplasia or the molecular inactivation mechanism of its loss in cases of advanced gastric cancer. In this study, we investigated the epigenetic inactivation of CDX1 by promoter methylation, as well as the functional link of CDX1 promoter methylation to the inflammatory NF-κB signaling pathway. We identified methylation-dependent NF-κB binding to the CDX1 promoter and quantified it using competitive electrophoretic mobility shift assays and chromatin immunoprecipitation. A methylated CDX1 promoter was associated with closed chromatin structure, reduced NF-κB binding, and transcriptional silencing. Along the gastritis-metaplasia-carcinoma sequence, we observed a biphasic pattern of tumor necrosis factor-α (TNF-α) protein expression and an inverse biphasic pattern of CDX1 promoter methylation; both are highly consistent with CDX1 protein expression. The stages of hyper-, hypo-, and hyper-methylation patterns of the CDX1 promoter were inversely correlated with the NF-κB signaling activity along this sequence. In conclusion, these functionally interacting events drive CDX1 expression and contribute to intestinal metaplasia, epithelial dedifferentiation, and carcinogenesis in the human stomach. PMID:22749770

Optimization of conditions for transient Agrobacterium-mediated gene expression assays in Arabidopsis.

PubMed

Kim, Mi Jung; Baek, Kon; Park, Chung-Mo

2009-08-01

Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The beta-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85-90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.

Characterization of Cer-1 cis-regulatory region during early Xenopus development.

PubMed

Silva, Ana Cristina; Filipe, Mário; Steinbeisser, Herbert; Belo, José António

2011-05-01

Cerberus-related molecules are well-known Wnt, Nodal, and BMP inhibitors that have been implicated in different processes including anterior–posterior patterning and left–right asymmetry. In both mouse and frog, two Cerberus-related genes have been isolated, mCer-1 and mCer-2, and Xcer and Xcoco, respectively. Until now, little is known about the mechanisms involved in their transcriptional regulation. Here, we report a heterologous analysis of the mouse Cerberus-1 gene upstream regulatory regions, responsible for its expression in the visceral endodermal cells. Our analysis showed that the consensus sequences for a TATA, CAAT, or GC boxes were absent but a TGTGG sequence was present at position -172 to -168 bp, relative to the ATG. Using a series of deletion constructs and transient expression in Xenopus embryos, we found that a fragment of 1.4 kb of Cer-1 promoter sequence could reproduce the endogenous expression pattern of Xenopus cerberus. A 0.7-kb mcer-1 upstream region was able to drive reporter expression to the involuting mesendodermal cells, while further deletions abolished reporter gene expression. Our results suggest that although no sequence similarity was found between mouse and Xenopus cerberus cis-regulatory regions, the signaling cascades regulating cerberus expression, during gastrulation, is conserved.

Patterning by heritage in mouse molar row development.

PubMed

Prochazka, Jan; Pantalacci, Sophie; Churava, Svatava; Rothova, Michaela; Lambert, Anne; Lesot, Hervé; Klein, Ophir; Peterka, Miroslav; Laudet, Vincent; Peterkova, Renata

2010-08-31

It is known from paleontology studies that two premolars have been lost during mouse evolution. During mouse mandible development, two bud-like structures transiently form that may represent rudimentary precursors of the lost premolars. However, the interpretation of these structures and their significance for mouse molar development are highly controversial because of a lack of molecular data. Here, we searched for typical tooth signaling centers in these two bud-like structures, and followed their fate using molecular markers, 3D reconstructions, and lineage tracing in vitro. Transient signaling centers were indeed found to be located at the tips of both the anterior and posterior rudimentary buds. These centers expressed a similar set of molecular markers as the "primary enamel knot" (pEK), the signaling center of the first molar (M1). These two transient signaling centers were sequentially patterned before and anterior to the M1 pEK. We also determined the dynamics of the M1 pEK, which, slightly later during development, spread up to the field formerly occupied by the posterior transient signaling center. It can be concluded that two rudimentary tooth buds initiate the sequential development of the mouse molars and these have previously been mistaken for early stages of M1 development. Although neither rudiment progresses to form an adult tooth, the posterior one merges with the adjacent M1, which may explain the anterior enlargement of the M1 during mouse family evolution. This study highlights how rudiments of lost structures can stay integrated and participate in morphogenesis of functional organs and help in understanding their evolution, as Darwin suspected long ago.

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss.

PubMed

Weld, R; Heinemann, J; Eady, C

2001-03-01

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells from Agrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.

Optogenetic mimicry of the transient activation of dopamine neurons by natural reward is sufficient for operant reinforcement.

PubMed

Kim, Kyung Man; Baratta, Michael V; Yang, Aimei; Lee, Doheon; Boyden, Edward S; Fiorillo, Christopher D

2012-01-01

Activation of dopamine receptors in forebrain regions, for minutes or longer, is known to be sufficient for positive reinforcement of stimuli and actions. However, the firing rate of dopamine neurons is increased for only about 200 milliseconds following natural reward events that are better than expected, a response which has been described as a "reward prediction error" (RPE). Although RPE drives reinforcement learning (RL) in computational models, it has not been possible to directly test whether the transient dopamine signal actually drives RL. Here we have performed optical stimulation of genetically targeted ventral tegmental area (VTA) dopamine neurons expressing Channelrhodopsin-2 (ChR2) in mice. We mimicked the transient activation of dopamine neurons that occurs in response to natural reward by applying a light pulse of 200 ms in VTA. When a single light pulse followed each self-initiated nose poke, it was sufficient in itself to cause operant reinforcement. Furthermore, when optical stimulation was delivered in separate sessions according to a predetermined pattern, it increased locomotion and contralateral rotations, behaviors that are known to result from activation of dopamine neurons. All three of the optically induced operant and locomotor behaviors were tightly correlated with the number of VTA dopamine neurons that expressed ChR2, providing additional evidence that the behavioral responses were caused by activation of dopamine neurons. These results provide strong evidence that the transient activation of dopamine neurons provides a functional reward signal that drives learning, in support of RL theories of dopamine function.

Steady-state pattern electroretinogram and short-duration transient visual evoked potentials in glaucomatous and healthy eyes.

PubMed

Amarasekera, Dilru C; Resende, Arthur F; Waisbourd, Michael; Puri, Sanjeev; Moster, Marlene R; Hark, Lisa A; Katz, L Jay; Fudemberg, Scott J; Mantravadi, Anand V

2018-01-01

This study evaluates two rapid electrophysiological glaucoma diagnostic tests that may add a functional perspective to glaucoma diagnosis. This study aimed to determine the ability of two office-based electrophysiological diagnostic tests, steady-state pattern electroretinogram and short-duration transient visual evoked potentials, to discern between glaucomatous and healthy eyes. This is a cross-sectional study in a hospital setting. Forty-one patients with glaucoma and 41 healthy volunteers participated in the study. Steady-state pattern electroretinogram and short-duration transient visual evoked potential testing was conducted in glaucomatous and healthy eyes. A 64-bar-size stimulus with both a low-contrast and high-contrast setting was used to compare steady-state pattern electroretinogram parameters in both groups. A low-contrast and high-contrast checkerboard stimulus was used to measure short-duration transient visual evoked potential parameters in both groups. Steady-state pattern electroretinogram parameters compared were MagnitudeD, MagnitudeD/Magnitude ratio, and the signal-to-noise ratio. Short-duration transient visual evoked potential parameters compared were amplitude and latency. MagnitudeD was significantly lower in glaucoma patients when using a low-contrast (P = 0.001) and high-contrast (P < 0.001) 64-bar-size steady-state pattern electroretinogram stimulus. MagnitudeD/Magnitude ratio and SNR were significantly lower in the glaucoma group when using a high-contrast 64-bar-size stimulus (P < 0.001 and P = 0.010, respectively). Short-duration transient visual evoked potential amplitude and latency were not significantly different between the two groups. Steady-state pattern electroretinogram was effectively able to discern between glaucomatous and healthy eyes. Steady-state pattern electroretinogram may thus have a role as a clinically useful electrophysiological diagnostic tool. © 2017 Royal Australian and New Zealand College of Ophthalmologists.

Cholinergic Plasticity of Oscillating Neuronal Assemblies in Mouse Hippocampal Slices

PubMed Central

Zylla, Maura M.; Zhang, Xiaomin; Reichinnek, Susanne; Draguhn, Andreas; Both, Martin

2013-01-01

The mammalian hippocampus expresses several types of network oscillations which entrain neurons into transiently stable assemblies. These groups of co-active neurons are believed to support the formation, consolidation and recall of context-dependent memories. Formation of new assemblies occurs during theta- and gamma-oscillations under conditions of high cholinergic activity. Memory consolidation is linked to sharp wave-ripple oscillations (SPW-R) during decreased cholinergic tone. We hypothesized that increased cholinergic tone supports plastic changes of assemblies while low cholinergic tone favors their stability. Coherent spatiotemporal network patterns were measured during SPW-R activity in mouse hippocampal slices. We compared neuronal activity within the oscillating assemblies before and after a transient phase of carbachol-induced gamma oscillations. Single units maintained their coupling to SPW-R throughout the experiment and could be re-identified after the transient phase of gamma oscillations. However, the frequency of SPW-R-related unit firing was enhanced after muscarinic stimulation. At the network level, these changes resulted in altered patterns of extracellularly recorded SPW-R waveforms. In contrast, recording of ongoing SPW-R activity without intermittent cholinergic stimulation revealed remarkably stable repetitive activation of assemblies. These results show that activation of cholinergic receptors induces plasticity at the level of oscillating hippocampal assemblies, in line with the different role of gamma- and SPW-R network activity for memory formation and –consolidation, respectively. PMID:24260462

Analysis of DNA-vaccinated fish reveals viral antigen in muscle, kidney, and thymus, and transient histopathologic changes

USGS Publications Warehouse

Garver, K.A.; Conway, C.M.; Elliott, D.G.; Kurath, G.

2005-01-01

A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was used in a systematic study to analyze vaccine tissue distribution, persistence, expression patterns, and histopathologic effects. Vaccine plasmid pIHNw-G, containing the gene for the viral glycoprotein, was detected immediately after intramuscular injection in all tissues analyzed, including blood, but at later time points was found primarily in muscle tissue, where it persisted to 90 days. Glycoprotein expression was detected in muscle, kidney, and thymus tissues, with levels peaking at 14 days and becoming undetectable by 28 days. Histologic examination revealed no vaccine-specific pathologic changes at the standard effective dose of 0.1 ??g DNA per fish, but at a high dose of 50 ??g an increased inflammatory response was evident. Transient damage associated with needle injection was localized in muscle tissue, but by 90 days after vaccination no damage was detected in any tissue, indicating the vaccine to be safe and well tolerated. ?? Springer Science+Business Media, Inc. 2005.

The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest.

PubMed

Gascoyne, Duncan M; Spearman, Hayley; Lyne, Linden; Puliyadi, Rathi; Perez-Alcantara, Marta; Coulton, Les; Fisher, Simon E; Croucher, Peter I; Banham, Alison H

2015-01-01

Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology.

The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest

PubMed Central

Gascoyne, Duncan M.; Spearman, Hayley; Lyne, Linden; Puliyadi, Rathi; Perez-Alcantara, Marta; Coulton, Les; Fisher, Simon E.; Croucher, Peter I.; Banham, Alison H.

2015-01-01

Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology. PMID:26034982

Antagonism between the transcription factors NANOG and OTX2 specifies rostral or caudal cell fate during neural patterning transition.

PubMed

Su, Zhenghui; Zhang, Yanqi; Liao, Baojian; Zhong, Xiaofen; Chen, Xin; Wang, Haitao; Guo, Yiping; Shan, Yongli; Wang, Lihui; Pan, Guangjin

2018-03-23

During neurogenesis, neural patterning is a critical step during which neural progenitor cells differentiate into neurons with distinct functions. However, the molecular determinants that regulate neural patterning remain poorly understood. Here we optimized the "dual SMAD inhibition" method to specifically promote differentiation of human pluripotent stem cells (hPSCs) into forebrain and hindbrain neural progenitor cells along the rostral-caudal axis. We report that neural patterning determination occurs at the very early stage in this differentiation. Undifferentiated hPSCs expressed basal levels of the transcription factor orthodenticle homeobox 2 (OTX2) that dominantly drove hPSCs into the "default" rostral fate at the beginning of differentiation. Inhibition of glycogen synthase kinase 3β (GSK3β) through CHIR99021 application sustained transient expression of the transcription factor NANOG at early differentiation stages through Wnt signaling. Wnt signaling and NANOG antagonized OTX2 and, in the later stages of differentiation, switched the default rostral cell fate to the caudal one. Our findings have uncovered a mutual antagonism between NANOG and OTX2 underlying cell fate decisions during neural patterning, critical for the regulation of early neural development in humans. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

[Cloning, subcellular localization, and heterologous expression of ApNAC1 gene from Andrographis paniculata].

PubMed

Wang, Jian; Qi, Meng-Die; Guo, Juan; Shen, Ye; Lin, Hui-Xin; Huang, Lu-Qi

2017-03-01

Andrographis paniculata is widely used as medicinal herb in China for a long time and andrographolide is its main medicinal constituent. To investigate the underlying andrographolide biosynthesis mechanisms, RNA-seq for A. paniculata leaves with MeJA treatment was performed. In A. paniculata transcriptomic data, the expression pattern of one member of NAC transcription factor family (ApNAC1) matched with andrographolide accumulation. The coding sequence of ApNAC1 was cloned by RT-PCR, and GenBank accession number was KY196416. The analysis of bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a predicted relative molecular weight of 35.9 kDa and isoelectric point of 6.14. To confirm the subcellular localization, ApNAC1-GFP was transiently expressed in A. paniculata protoplast. The results indicated that ApNAC1 is a nucleus-localized protein. The analysis of real-time quantitative PCR revealed that ApNAC1 gene predominantly expresses in leaves. Compared with control sample, its expression abundance sharply increased with methyl jasmonate treatment. Based on its expression pattern, ApNAC1 gene might involve in andrographolide biosynthesis. ApNAC1 was heterologously expressed in Escherichia coli and recombinant protein was purified by Ni-NTA agarose. Further study will help us to understand the function of ApNAC1 in andrographolide biosynthesis. Copyright© by the Chinese Pharmaceutical Association.

The murine homeobox gene Msx-3 shows highly restricted expression in the developing neural tube.

PubMed

Shimeld, S M; McKay, I J; Sharpe, P T

1996-04-01

The mouse homeobox-genes Msx-1 and Msx-2 are expressed in several areas of the developing embryo, including the neural tube, neural crest, facial processes and limb buds. Here we report the characterisation of a third mouse Msx gene, which we designate Msx-3. The embryonic expression of Msx-3 was found to differ from that of Msx-1 and -2 in that it was confined to the dorsal neural tube. In embryos with 5-8 somites a segmental pattern of expression was observed in the hindbrain, with rhombomeres 3 and 5 lacking Msx-3 while other rhombomeres expressed Msx-3. This pattern was transient, however, such that in embryos with 18 or more somites expression was continuous throughout the dorsal hindbrain and anterior dorsal spinal cord. Differentiation of dorsal cell types in the neural tube can be induced by addition of members of the Tgf-beta family. Additionally, Msx-1 and -2 have been shown to be activated by addition of the Tgf-beta family member Bmp-4. To determine if Bmp-4 could activate Msx-3, we incubated embryonic hindbrain explants with exogenous Bmp-4. The dorsal expression of Msx-3 was seen to expand into more ventral regions of the neurectoderm in Bmp-4-treated cultures, implying that Bmp-4 may be able to mimic an in vivo signal that induces Msx-3.

Temporal, spatial, and phenotypical changes of PDGFRα expressing fibroblasts during late lung development.

PubMed

Endale, Mehari; Ahlfeld, Shawn; Bao, Erik; Chen, Xiaoting; Green, Jenna; Bess, Zach; Weirauch, Matthew T; Xu, Yan; Perl, Anne Karina

2017-05-15

Many studies have investigated the source and role of epithelial progenitors during lung development; such information is limited for fibroblast populations and their complex role in the developing lung. In this study, we characterized the spatial location, mRNA expression and Immunophenotyping of PDGFRα + fibroblasts during sacculation and alveolarization. Confocal microscopy identified spatial association of PDGFRα expressing fibroblasts with proximal epithelial cells of the branching bronchioles and the dilating acinar tubules at E16.5; with distal terminal saccules at E18.5; and with alveolar epithelial cells at PN7 and PN28. Immunohistochemistry for alpha smooth muscle actin revealed that PDGFRα + fibroblasts contribute to proximal peribronchiolar smooth muscle at E16.5 and to transient distal alveolar myofibroblasts at PN7. Time series RNA-Seq analyses of PDGFRα + fibroblasts identified differentially expressed genes that, based on gene expression similarity were clustered into 7 major gene expression profile patterns. The presence of myofibroblast and smooth muscle precursors at E16.5 and PN7 was reflected by a two-peak gene expression profile on these days and gene ontology enrichment in muscle contraction. Additional molecular and functional differences between peribronchiolar smooth muscle cells at E16.5 and transient intraseptal myofibroblasts at PN7 were suggested by a single peak in gene expression at PN7 with functional enrichment in cell projection and muscle cell differentiation. Immunophenotyping of subsets of PDGFRα + fibroblasts by flow cytometry confirmed the predicted increase in proliferation at E16.5 and PN7, and identified subsets of CD29 + myofibroblasts and CD34 + lipofibroblasts. These data can be further mined to develop novel hypotheses and valuable understanding of the molecular and cellular basis of alveolarization. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Temporal, spatial, and phenotypical changes of PDGFRα expressing fibroblasts during late lung development☆

PubMed Central

Endale, Mehari; Ahlfeld, Shawn; Bao, Erik; Chen, Xiaoting; Green, Jenna; Bess, Zach; Weirauch, Matthew T.; Xu, Yan; Perl, Anne Karina

2017-01-01

Many studies have investigated the source and role of epithelial progenitors during lung development; such information is limited for fibroblast populations and their complex role in the developing lung. In this study, we characterized the spatial location, mRNA expression and Immunophenotyping of PDGFRα+ fibroblasts during sacculation and alveolarization. Confocal microscopy identified spatial association of PDGFRα expressing fibroblasts with proximal epithelial cells of the branching bronchioles and the dilating acinar tubules at E16.5; with distal terminal saccules at E18.5; and with alveolar epithelial cells at PN7 and PN28. Immunohistochemistry for alpha smooth muscle actin revealed that PDGFRα+ fibroblasts contribute to proximal peribronchiolar smooth muscle at E16.5 and to transient distal alveolar myofibroblasts at PN7. Time series RNA-Seq analyses of PDGFRα+ fibroblasts identified differentially expressed genes that, based on gene expression similarity were clustered into 7 major gene expression profile patterns. The presence of myofibroblast and smooth muscle precursors at E16.5 and PN7 was reflected by a two-peak gene expression profile on these days and gene ontology enrichment in muscle contraction. Additional molecular and functional differences between peribronchiolar smooth muscle cells at E16.5 and transient intraseptal myofibroblasts at PN7 were suggested by a single peak in gene expression at PN7 with functional enrichment in cell projection and muscle cell differentiation. Immunophenotyping of subsets of PDGFRα+ fibroblasts by flow cytometry confirmed the predicted increase in proliferation at E16.5 and PN7, and identified subsets of CD29+ myofibroblasts and CD34+ lipofibroblasts. These data can be further mined to develop novel hypotheses and valuable understanding of the molecular and cellular basis of alveolarization. PMID:28408205

Neurogenesis and ontogeny of specific cell phenotypes within the hamster suprachiasmatic nucleus.

PubMed

Antle, Michael C; LeSauter, Joseph; Silver, Rae

2005-06-09

The hamster suprachiasmatic nucleus (SCN) is anatomically and functionally heterogeneous. A group of cells in the SCN shell, delineated by vasopressin-ergic neurons, are rhythmic with respect to Period gene expression and electrical activity but do not receive direct retinal input. In contrast, some cells in the SCN core, marked by neurons containing calbindin-D28k, gastrin-releasing peptide (GRP), substance P (SP), and vasoactive intestinal polypeptide (VIP), are not rhythmic with respect to Period gene expression and electrical activity but do receive direct retinal input. Examination of the timing of neurogenesis using bromodeoxyuridine indicates that SCN cells are born between embryonic day 9.5 and 12.5. Calbindin, GRP, substance P, and VIP cells are born only during early SCN neurogenesis, between embryonic days 9.5-11.0. Vasopressin cells are born over the whole period of SCN neurogenesis, appearing as late as embryonic day 12.5. Examination of the ontogeny of peptide expression in these cell types reveals transient expression of calbindin in a cluster of dorsolateral SCN cells on postnatal days 1-2. The adult pattern of calbindin expression is detected in a different ventrolateral cell cluster starting on postnatal day 2. GRP and SP expression appear on postnatal day 8 and 10, respectively, after the retinohypothalamic tract has innervated the SCN. In summary, the present study describes the ontogeny-specific peptidergic phenotypes in the SCN and compares these developmental patterns to previously identified patterns in the appearance of circadian functions. These comparisons suggest the possibility that these coincident appearances may be causally related, with the direction of causation to be determined.

Potato NPH3/RPT2-Like Protein StNRL1, Targeted by a Phytophthora infestans RXLR Effector, Is a Susceptibility Factor.

PubMed

Yang, Lina; McLellan, Hazel; Naqvi, Shaista; He, Qin; Boevink, Petra C; Armstrong, Miles; Giuliani, Licida M; Zhang, Wei; Tian, Zhendong; Zhan, Jiasui; Gilroy, Eleanor M; Birch, Paul R J

2016-05-01

Plant pathogens deliver effectors to manipulate host processes. We know little about how fungal and oomycete effectors target host proteins to promote susceptibility, yet such knowledge is vital to understand crop disease. We show that either transient expression in Nicotiana benthamiana, or stable transgenic expression in potato (Solanum tuberosum), of the Phytophthora infestans RXLR effector Pi02860 enhances leaf colonization by the pathogen. Expression of Pi02860 also attenuates cell death triggered by the P. infestans microbe-associated molecular pattern INF1, indicating that the effector suppresses pattern-triggered immunity. However, the effector does not attenuate cell death triggered by Cf4/Avr4 coexpression, showing that it does not suppress all cell death activated by cell surface receptors. Pi02860 interacts in yeast two-hybrid assays with potato NPH3/RPT2-LIKE1 (NRL1), a predicted CULLIN3-associated ubiquitin E3 ligase. Interaction of Pi02860 in planta was confirmed by coimmunoprecipitation and bimolecular fluorescence complementation assays. Virus-induced gene silencing of NRL1 in N. benthamiana resulted in reduced P. infestans colonization and accelerated INF1-mediated cell death, indicating that this host protein acts as a negative regulator of immunity. Moreover, whereas NRL1 virus-induced gene silencing had no effect on the ability of the P. infestans effector Avr3a to suppress INF1-mediated cell death, such suppression by Pi02860 was significantly attenuated, indicating that this activity of Pi02860 is mediated by NRL1. Transient overexpression of NRL1 resulted in the suppression of INF1-mediated cell death and enhanced P. infestans leaf colonization, demonstrating that NRL1 acts as a susceptibility factor to promote late blight disease. © 2016 American Society of Plant Biologists. All Rights Reserved.

Induction of innate immune responses by flagellin from the intracellular bacterium, 'Candidatus Liberibacter solanacearum'.

PubMed

Hao, Guixia; Pitino, Marco; Ding, Fang; Lin, Hong; Stover, Ed; Duan, Yongping

2014-08-05

'Candidatus Liberibacter solanacearum' (Lso) is a phloem-limited alphaproteobacterium associated with the devastating zebra chip disease of potato (Solanum tuberosum). Like other members of Liberibacter, Lso-ZC1 encodes a flagellin domain-containing protein (Fla Lso ) with a conserved 22 amino-acid peptide (flg22 Lso ). To understand the innate immune responses triggered by this unculturable intracellular bacterium, we studied the pathogen-associated molecular patterns (PAMPs) that triggered immunity in Nicotiana benthamiana, using the flg22 Lso peptide and the full length fla Lso gene. Our results showed that the expression of fla Lso via Agrobacterium-mediated transient expression induced a slow necrotic cell death in the inoculated leaves of N. benthamiana, which was coupled with a burst of reactive oxygen species (ROS) production. Moreover, the expression of several representative genes involved in innate immunity was transiently up-regulated by the flg22 Lso in N. benthamiana. The Fla Lso , however, induced stronger up-regulation of these representative genes compared to the flg22 Lso , especially that of flagellin receptor FLAGELLIN SENSING2 (FLS2) and respiratory burst oxidase (RbohB) in N. benthamiana. Although neither cell death nor ROS were induced by the synthetic flg22 Lso , a weak callose deposition was observed in infiltrated leaves of tobacco, tomato, and potato plants. The flagellin of Lso and its functional domain, flg22 Lso share characteristics of pathogen-associated molecular patterns, and trigger unique innate immune responses in N. benthamiana. Slow and weak activation of the innate immune response in host plants by the flagellin of Lso may reflect the nature of its intracellular life cycle. Our findings provide new insights into the role of the Lso flagellin in the development of potato zebra chip disease and potential application in breeding for resistance.

Mature Luffa Leaves (Luffa cylindrica L.) as a Tool for Gene Expression Analysis by Agroinfiltration

PubMed Central

Błażejewska, Kamila; Kapusta, Małgorzata; Zielińska, Elżbieta; Tukaj, Zbigniew; Chincinska, Izabela A.

2017-01-01

We exploited the potential of cucurbits for ectopic gene expression. Agroinfiltration is a simple and commonly used method to obtain transient expression of foreign genes in plants. In contrast to in vitro transformation techniques, agroinfiltration can be used for genetic modification of mature plant tissues. Although the cucurbits are commonly used as model plants for molecular biology and biotechnology studies, to date there are no literature sources on the possibility of transient gene expression in mature cucurbit tissues. Our research has shown that mature leaves of Luffa cylindrica L. (luffa), in contrast to other cucurbit species, can be successfully transiently transformed with Agrobacterium tumefaciens. We efficiently transformed luffa leaves with a reporter gene encoding β-glucuronidase (GUS). The GUS activity in transiently transformed leaf tissues was detected within 24 h after the infiltration with bacteria. Additionally, we have shown that the activity of a transiently expressed the GUS gene can be monitored directly in the EDTA-exudates collected from the cut petioles of the agroinfiltrated leaves. The results suggest that luffa leaves can be useful as a plant expression system for studies of physiological and biochemical processes in cucurbits. PMID:28270826

Impaired glucose homeostasis in transgenic mice expressing the human transient neonatal diabetes mellitus locus, TNDM

PubMed Central

Ma, Dan; Shield, Julian P.H.; Dean, Wendy; Leclerc, Isabelle; Knauf, Claude; Burcelin, Rémy; Rutter, Guy A.; Kelsey, Gavin

2004-01-01

Transient neonatal diabetes mellitus (TNDM) is a rare inherited diabetic syndrome apparent in the first weeks of life and again during early adulthood. The relative contributions of reduced islet β cell number and impaired β cell function to the observed hypoinsulinemia are unclear. The inheritance pattern of this imprinted disorder implicates overexpression of one or both genes within the TNDM locus: ZAC, which encodes a proapoptotic zinc finger protein, and HYMAI, which encodes an untranslated mRNA. To investigate the consequences for pancreatic function, we have developed a high-copy transgenic mouse line, TNDM29, carrying the human TNDM locus. TNDM29 neonates display hyperglycemia, and older adults, impaired glucose tolerance. Neonatal hyperglycemia occurs only on paternal transmission, analogous to paternal dependence of TNDM in humans. Embryonic pancreata of TNDM29 mice showed reductions in expression of endocrine differentiation factors and numbers of insulin-staining structures. By contrast, β cell mass was normal or elevated at all postnatal stages, whereas pancreatic insulin content in neonates and peak serum insulin levels after glucose infusion in adults were reduced. Expression of human ZAC and HYMAI in these transgenic mice thus recapitulates key features of TNDM and implicates impaired development of the endocrine pancreas and β cell function in disease pathogenesis. PMID:15286800

Prediction of Bacillus weihenstephanensis acid resistance: the use of gene expression patterns to select potential biomarkers.

PubMed

Desriac, N; Postollec, F; Coroller, L; Sohier, D; Abee, T; den Besten, H M W

2013-10-01

Exposure to mild stress conditions can activate stress adaptation mechanisms and provide cross-resistance towards otherwise lethal stresses. In this study, an approach was followed to select molecular biomarkers (quantitative gene expressions) to predict induced acid resistance after exposure to various mild stresses, i.e. exposure to sublethal concentrations of salt, acid and hydrogen peroxide during 5 min to 60 min. Gene expression patterns of unstressed and mildly stressed cells of Bacillus weihenstephanensis were correlated to their acid resistance (3D value) which was estimated after exposure to lethal acid conditions. Among the twenty-nine candidate biomarkers, 12 genes showed expression patterns that were correlated either linearly or non-linearly to acid resistance, while for the 17 other genes the correlation remains to be determined. The selected genes represented two types of biomarkers, (i) four direct biomarker genes (lexA, spxA, narL, bkdR) for which expression patterns upon mild stress treatment were linearly correlated to induced acid resistance; and (ii) nine long-acting biomarker genes (spxA, BcerKBAB4_0325, katA, trxB, codY, lacI, BcerKBAB4_1716, BcerKBAB4_2108, relA) which were transiently up-regulated during mild stress exposure and correlated to increased acid resistance over time. Our results highlight that mild stress induced transcripts can be linearly or non-linearly correlated to induced acid resistance and both approaches can be used to find relevant biomarkers. This quantitative and systematic approach opens avenues to select cellular biomarkers that could be incremented in mathematical models to predict microbial behaviour. Copyright © 2013 Elsevier B.V. All rights reserved.

Bacterial Competition Reveals Differential Regulation of the pks Genes by Bacillus subtilis

PubMed Central

Vargas-Bautista, Carol; Rahlwes, Kathryn

2014-01-01

Bacillus subtilis is adaptable to many environments in part due to its ability to produce a broad range of bioactive compounds. One such compound, bacillaene, is a linear polyketide/nonribosomal peptide. The pks genes encode the enzymatic megacomplex that synthesizes bacillaene. The majority of pks genes appear to be organized as a giant operon (>74 kb from pksC-pksR). In previous work (P. D. Straight, M. A. Fischbach, C. T. Walsh, D. Z. Rudner, and R. Kolter, Proc. Natl. Acad. Sci. U. S. A. 104:305–310, 2007, doi:10.1073/pnas.0609073103), a deletion of the pks operon in B. subtilis was found to induce prodiginine production by Streptomyces coelicolor. Here, colonies of wild-type B. subtilis formed a spreading population that induced prodiginine production from Streptomyces lividans, suggesting differential regulation of pks genes and, as a result, bacillaene. While the parent colony showed widespread induction of pks expression among cells in the population, we found the spreading cells uniformly and transiently repressed the expression of the pks genes. To identify regulators that control pks genes, we first determined the pattern of pks gene expression in liquid culture. We next identified mutations in regulatory genes that disrupted the wild-type pattern of pks gene expression. We found that expression of the pks genes requires the master regulator of development, Spo0A, through its repression of AbrB and the stationary-phase regulator, CodY. Deletions of degU, comA, and scoC had moderate effects, disrupting the timing and level of pks gene expression. The observed patterns of expression suggest that complex regulation of bacillaene and other antibiotics optimizes competitive fitness for B. subtilis. PMID:24187085

Bacterial competition reveals differential regulation of the pks genes by Bacillus subtilis.

PubMed

Vargas-Bautista, Carol; Rahlwes, Kathryn; Straight, Paul

2014-02-01

Bacillus subtilis is adaptable to many environments in part due to its ability to produce a broad range of bioactive compounds. One such compound, bacillaene, is a linear polyketide/nonribosomal peptide. The pks genes encode the enzymatic megacomplex that synthesizes bacillaene. The majority of pks genes appear to be organized as a giant operon (>74 kb from pksC-pksR). In previous work (P. D. Straight, M. A. Fischbach, C. T. Walsh, D. Z. Rudner, and R. Kolter, Proc. Natl. Acad. Sci. U. S. A. 104:305-310, 2007, doi:10.1073/pnas.0609073103), a deletion of the pks operon in B. subtilis was found to induce prodiginine production by Streptomyces coelicolor. Here, colonies of wild-type B. subtilis formed a spreading population that induced prodiginine production from Streptomyces lividans, suggesting differential regulation of pks genes and, as a result, bacillaene. While the parent colony showed widespread induction of pks expression among cells in the population, we found the spreading cells uniformly and transiently repressed the expression of the pks genes. To identify regulators that control pks genes, we first determined the pattern of pks gene expression in liquid culture. We next identified mutations in regulatory genes that disrupted the wild-type pattern of pks gene expression. We found that expression of the pks genes requires the master regulator of development, Spo0A, through its repression of AbrB and the stationary-phase regulator, CodY. Deletions of degU, comA, and scoC had moderate effects, disrupting the timing and level of pks gene expression. The observed patterns of expression suggest that complex regulation of bacillaene and other antibiotics optimizes competitive fitness for B. subtilis.

Complex Patterns of Altered MicroRNA Expression during the Adenoma-Adenocarcinoma Sequence for Microsatellite-Stable Colorectal Cancer

PubMed Central

Bartley, Angela N.; Yao, Hui; Barkoh, Bedia A.; Ivan, Cristina; Mishra, Bal M.; Rashid, Asif; Calin, George A.; Luthra, Rajyalakshmi; Hamilton, Stanley R.

2012-01-01

Purpose MicroRNAs are short noncoding RNAs that regulate gene expression and are over- or under-expressed in most tumors, including colorectal adenocarcinoma. MicroRNAs are potential biomarkers and therapeutic targets and agents, but limited information on microRNAome alterations during progression in the well-known adenoma-adenocarcinoma sequence is available to guide their usage. Experimental Design We profiled 866 human microRNAs by microarray analysis in 69 matched specimens of microsatellite-stable adenocarcinomas, adjoining precursor adenomas including areas of high- and low-grade dysplasia, and nonneoplastic mucosa. Results We found 230 microRNAs that were significantly differentially expressed during progression, including 19 not reported previously. Altered microRNAs clustered into two major patterns of early (type I) and late (type II) differential expression. The largest number (n = 108) was altered at the earliest step from mucosa to low-grade dysplasia (subtype IA) prior to major nuclear localization of β-catenin, including 36 microRNAs that had persistent differential expression throughout the entire sequence to adenocarcinoma. Twenty microRNAs were intermittently altered (subtype IB), and six were transiently altered (subtype IC). In contrast, 33 microRNAs were altered late in high-grade dysplasia and adenocarcinoma (subtype IIA), and 63 in adenocarcinoma only (subtype IIB). Predicted targets in 12 molecular pathways were identified for highly altered microRNAs, including the Wnt signaling pathway leading to low-grade dysplasia. β-catenin expression correlated with downregulated microRNAs. Conclusions Our findings suggest that numerous microRNAs play roles in the sequence of molecular events, especially early events, resulting in colorectal adenocarcinoma. The temporal patterns and complexity of microRNAome alterations during progression will influence the efficacy of microRNAs for clinical purposes. PMID:21948089

Characterization of the Martian magnetic topology response to extreme solar transient events with MGS data

NASA Astrophysics Data System (ADS)

Xu, S.; Curry, S.; Mitchell, D. L.; Luhmann, J. G.; Lillis, R. J.; Dong, C.

2017-12-01

Characterizing how the solar cycle affects the physics of the Mars-solar wind interaction can improve our understanding of Mars' atmospheric evolution and the plasma environment at Mars. In particular, solar transient events such as Interplanetary Coronal Mass Ejections (ICMEs) and Stream Interaction Regions (SIRs) significantly change the solar-wind interaction, including the magnetic topology and ion acceleration. However, both the Mars Express and Mars Atmosphere Volatile EvolutioN (MAVEN) missions have encountered relatively few extreme solar transient events due to the recent low solar activity (2004-2017). In contrast, Mars Global Surveyor (MGS) was operating during a relatively active solar maximum (1999-2003). Based on new results from MAVEN, this study reanalyzes MGS data to better understand how the Martian plasma environment responds to extreme solar events. In particular, we aim to investigate how the magnetic topology during these extreme events differs from the topology during quiet times. We conduct orbit comparisons of the magnetic topology inferred from MGS electron pitch angle distributions during quiet periods and extreme events to determine how the open and closed field patterns respond to extreme events.

Expression of Histone Deacetylases in Cellular Compartments of the Mouse Brain and the Effects of Ischemia

PubMed Central

Bachleda, Amelia; Morrison, Richard S.; Murphy, Sean P.

2011-01-01

Drugs that inhibit specific histone deacetylase (HDAC) activities have enormous potential in preventing the consequences of acute injury to the nervous system and in allaying neurodegeneration. However, very little is known about the expression pattern of the HDACs in the central nervous system (CNS). Identifying the cell types that express HDACs in the CNS is important for determining therapeutic targets for HDAC inhibitors and evaluating potential side effects. We characterized the cellular expression of HDACs 1–3, and HDACs 4 and 6, in the adult mouse brain in the cingulate cortex, parietal cortex, dentate gyrus, and CA1 regions of the hippocampus and subcortical white matter. Expression of class I HDACs showed a cell-and region-specific pattern. Transient focal ischemia induced by temporary middle cerebral artery occlusion, or global ischemia induced by in vitro oxygen–glucose deprivation, altered the extent of HDAC expression in a region- and cell-specific manner. The pan-HDAC inhibitor, SAHA, reduced ischemia-induced alterations in HDACs. The results suggest that in addition to promoting epigenetic changes in transcriptional activity in the nucleus of neurons and glia, HDACs may also have non-transcriptional actions in axons and the distant processes of glial cells and may significantly modulate the response to injury in a cell- and region-specific manner. PMID:21966324

Expression patterns of epiplakin1 in pancreas, pancreatic cancer and regenerating pancreas.

PubMed

Yoshida, Tetsu; Shiraki, Nobuaki; Baba, Hideo; Goto, Mizuki; Fujiwara, Sakuhei; Kume, Kazuhiko; Kume, Shoen

2008-07-01

Epiplakin1 (Eppk1) is a plakin family gene with its function remains largely unknown, although the plakin genes are known to function in interconnecting cytoskeletal filaments and anchoring them at plasma membrane-associated adhesive junction. Here we analyzed the expression patterns of Eppk1 in the developing and adult pancreas in the mice. In the embryonic pancreas, Eppk1+/Pdx1+ and Eppk1+/Sox9+ pancreatic progenitor cells were observed in early pancreatic epithelium. Since Pdx1 expression overlapped with that of Sox9 at this stage, these multipotent progenitor cells are Eppk1+/Pdx1+/Sox9+ cells. Then Eppk1 expression becomes confined to Ngn3+ or Sox9+ endocrine progenitor cells, and p48+ exocrine progenitor cells, and then restricted to the duct cells and a cells at birth. In the adult pancreas, Eppk1 is expressed in centroacinar cells (CACs) and in duct cells. Eppk1 is observed in pancreatic intraepithelial neoplasia (PanIN), previously identified as pancreatic ductal adenocarcinoma (PDAC) precursor lesions. In addition, the expansion of Eppk1-positive cells occurs in a caerulein-induced acute pancreatitis, an acinar cell regeneration model. Furthermore, in the partial pancreatectomy (Px) regeneration model using mice, Eppk1 is expressed in "ducts in foci", a tubular structure transiently induced. These results suggest that Eppk1 serves as a useful marker for detecting pancreatic progenitor cells in developing and regenerating pancreas.

Myb14, a direct activator of STS, is associated with resveratrol content variation in berry skin in two grape cultivars.

PubMed

Fang, Linchuan; Hou, Yanlin; Wang, Lijun; Xin, Haiping; Wang, Nian; Li, Shaohua

2014-10-01

High and low resveratrol (Res) contents in two cultivars are correlated with the expression abundance of Myb14 , which could directly activate transcriptional expression of stilbene synthase gene ( STS ). Resveratrol (3,5,4'-trihydroxystilbene) is one of the natural polyphenols produced by secondary metabolism in some plants. Stilbene synthase (STS) is the key enzyme for the final step of precursor formation of resveratrol (Res) in grapevines. In this study, we found that Res contents in ripe berry skin were completely different in two grape cultivars, namely, 'Z168' (Vitis monticola × Vitis riparia) with high-Res and 'Jingzaojing' (Vitis vinifera) with low-Res. Moreover, the level of expression of STS gene was higher in the ripe berry skin of 'Z168' than in that of 'Jingzaojing'. To further investigate the underlying mechanisms, we conducted a co-expression analysis through transcriptomic data. We confirmed that Myb14, an R2R3 Myb transcription factor, is the direct regulator of STS by binding to Box-L5 motif. Moreover, the expression pattern of Myb14 is associated with the variation of Res content. To test this prediction, we conducted a number of experiments in vivo and in vitro. The expression patterns of Myb14 and STS in grapevine leaves were identical under a series of stimulus. Myb14 showed higher expression in the ripe berry skin of 'Z168' than in that of 'Jingzaojing'. Yeast one-hybrid assay indicated that grapevine Myb14 could interact with the promoter of STS in vitro, and the transient overexpression of Myb14 promoted the expression of STS. Furthermore, co-expressing 35S::Myb14 in transgenic Arabidopsis could activate GUS expression promoted by STS promoter. Thus, Myb14 is the direct activator of STS, and its expression pattern is associated with Res content variation in grapes.

Metastable dynamical patterns and their stabilization in arrays of bidirectionally coupled sigmoidal neurons

NASA Astrophysics Data System (ADS)

Horikawa, Yo

2013-12-01

Transient patterns in a bistable ring of bidirectionally coupled sigmoidal neurons were studied. When the system had a pair of spatially uniform steady solutions, the instability of unstable spatially nonuniform steady solutions decreased exponentially with the number of neurons because of the symmetry of the system. As a result, transient spatially nonuniform patterns showed dynamical metastability: Their duration increased exponentially with the number of neurons and the duration of randomly generated patterns obeyed a power-law distribution. However, these metastable dynamical patterns were easily stabilized in the presence of small variations in coupling strength. Metastable rotating waves and their pinning in the presence of asymmetry in the direction of coupling and the disappearance of metastable dynamical patterns due to asymmetry in the output function of a neuron were also examined. Further, in a two-dimensional array of neurons with nearest-neighbor coupling, intrinsically one-dimensional patterns were dominant in transients, and self-excitation in these neurons affected the metastable dynamical patterns.

Developmental expression and function analysis of protein tyrosine phosphatase receptor type D in oligodendrocyte myelination

PubMed Central

Zhu, Qiang; Tan, Zhou; Zhao, Shufang; Huang, Hao; Zhao, Xiaofeng; Hu, Xuemei; Zhang, Yiping; Shields, Christopher B; Uetani, Noriko; Qiu, Mengsheng

2015-01-01

Receptor protein tyrosine phosphatases (RPTPs) are extensively expressed in the central nervous system (CNS), and have distinct spatial and temporal patterns in different cell types during development. Previous studies have demonstrated possible roles for RPTPs in axon outgrowth, guidance, and synaptogenesis. In the present study, our results revealed that protein tyrosine phosphatase, receptor type D (PTPRD) was initially expressed in mature neurons in embryonic CNS, and later in oligodendroglial cells at postnatal stages when oligodendrocyte undergo active axonal myelination process. In PTPRD mutants, oligodendrocyte differentiation was normal and a transient myelination delay occurred at early postnatal stages, indicating the contribution of PTPRD to the initiation of axonal myelination. Our results also showed that the remyelination process was not affected in the absence of PTPRD function after a cuprizone-induced demyelination in adult animals. PMID:26341907

Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood.

PubMed

Souza, K de Picoli; Nunes, M T

2014-08-01

Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g) and triiodothyronine (0.5-50 µg/100 g) for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P) days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold). Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60%) ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold), and decreased heart rate (5%), fast muscle myoglobin mRNA (30%) and body weight (20%) in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30%) and body weight (14%). These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood.

Rapid activation of ERK1/2 and AKT in human breast cancer cells by cadmium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Zhiwei; Yu Xinyuan; Shaikh, Zahir A.

2008-05-01

Cadmium (Cd), an endocrine disruptor, can induce a variety of signaling events including the activation of ERK1/2 and AKT. In this study, the involvement of estrogen receptors (ER) in these events was evaluated in three human breast caner cell lines, MCF-7, MDA-MB-231, and SK-BR-3. The Cd-induced signal activation patterns in the three cell lines mimicked those exhibited in response to 17{beta}-estradiol. Specifically, treatment of MCF-7 cells, that express ER{alpha}, ER{beta} and GPR30, to 0.5-10 {mu}M Cd for only 2.5 min resulted in transient phosphorylation of ERK1/2. Cd also triggered a gradual increase and sustained activation of AKT during the 60more » min treatment period. In SK-BR-3 cells, that express only GPR30, Cd also caused a transient activation of ERK1/2, but not of AKT. In contrast, in MDA-MB-231 cells, that express only ER{beta}, Cd was unable to cause rapid activation of either ERK1/2 or AKT. A transient phosphorylation of ER{alpha} was also observed within 2.5 min of Cd exposure in the MCF-7 cells. While the estrogen receptor antagonist, ICI 182,780, did not prevent the effect of Cd on these signals, specific siRNA against hER{alpha} significantly reduced Cd-induced activation of ERK1/2 and completely blocked the activation of AKT. It is concluded that Cd, like estradiol, can cause rapid activation of ERK1/2 and AKT and that these signaling events are mediated by possible interaction with membrane ER{alpha} and GPR30, but not ER{beta}.« less

Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood

PubMed Central

de Picoli Souza, K.; Nunes, M.T.

2014-01-01

Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g) and triiodothyronine (0.5-50 µg/100 g) for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P) days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold). Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60%) ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold), and decreased heart rate (5%), fast muscle myoglobin mRNA (30%) and body weight (20%) in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30%) and body weight (14%). These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood. PMID:25098716

Hyperexcitability of bladder afferent neurons associated with reduction of Kv1.4 α-subunit in rats with spinal cord injury.

PubMed

Takahashi, Ryosuke; Yoshizawa, Tsuyoshi; Yunoki, Takakazu; Tyagi, Pradeep; Naito, Seiji; de Groat, William C; Yoshimura, Naoki

2013-12-01

To clarify the functional and molecular mechanisms inducing hyperexcitability of C-fiber bladder afferent pathways after spinal cord injury we examined changes in the electrophysiological properties of bladder afferent neurons, focusing especially on voltage-gated K channels. Freshly dissociated L6-S1 dorsal root ganglion neurons were prepared from female spinal intact and spinal transected (T9-T10 transection) Sprague Dawley® rats. Whole cell patch clamp recordings were performed on individual bladder afferent neurons. Kv1.2 and Kv1.4 α-subunit expression levels were also evaluated by immunohistochemical and real-time polymerase chain reaction methods. Capsaicin sensitive bladder afferent neurons from spinal transected rats showed increased cell excitability, as evidenced by lower spike activation thresholds and a tonic firing pattern. The peak density of transient A-type K+ currents in capsaicin sensitive bladder afferent neurons from spinal transected rats was significantly less than that from spinal intact rats. Also, the KA current inactivation curve was displaced to more hyperpolarized levels after spinal transection. The protein and mRNA expression of Kv1.4 α-subunits, which can form transient A-type K+ channels, was decreased in bladder afferent neurons after spinal transection. Results indicate that the excitability of capsaicin sensitive C-fiber bladder afferent neurons is increased in association with reductions in transient A-type K+ current density and Kv1.4 α-subunit expression in injured rats. Thus, the Kv1.4 α-subunit could be a molecular target for treating overactive bladder due to neurogenic detrusor overactivity. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Mechanisms of Transient Signaling via Short and Long Prolactin Receptor Isoforms in Female and Male Sensory Neurons*

PubMed Central

Belugin, Sergei; Diogenes, Anibal R.; Patil, Mayur J.; Ginsburg, Erika; Henry, Michael A.; Akopian, Armen N.

2013-01-01

Prolactin (PRL) regulates activity of nociceptors and causes hyperalgesia in pain conditions. PRL enhances nociceptive responses by rapidly modulating channels in nociceptors. The molecular mechanisms underlying PRL-induced transient signaling in neurons are not well understood. Here we use a variety of cell biology and pharmacological approaches to show that PRL transiently enhanced capsaicin-evoked responses involve protein kinase C ϵ (PKCϵ) or phosphatidylinositol 3-kinase (PI3K) pathways in female rat trigeminal (TG) neurons. We next reconstituted PRL-induced signaling in a heterologous expression system and TG neurons from PRL receptor (PRLR)-null mutant mice by expressing rat PRLR-long isoform (PRLR-L), PRLR-short isoform (PRLR-S), or a mix of both. Results show that PRLR-S, but not PRLR-L, is capable of mediating PRL-induced transient enhancement of capsaicin responses in both male and female TG neurons. However, co-expression of PRLR-L with PRLR-S (1:1 ratio) leads to the inhibition of the transient PRL actions. Co-expression of PRLR-L deletion mutants with PRLR-S indicated that the cytoplasmic site adjacent to the trans-membrane domain of PRLR-L was responsible for inhibitory effects of PRLR-L. Furthermore, in situ hybridization and immunohistochemistry data indicate that in normal conditions, PRLR-L is expressed mainly in glia with little expression in rat sensory neurons (3–5%) and human nerves. The predominant PRLR form in TG neurons/nerves from rats and humans is PRLR-S. Altogether, PRL-induced transient signaling in sensory neurons is governed by PI3K or PKCϵ, mediated via the PRLR-S isoform, and transient effects mediated by PRLR-S are inhibited by presence of PRLR-L in these cells. PMID:24142695

Mechanisms of transient signaling via short and long prolactin receptor isoforms in female and male sensory neurons.

PubMed

Belugin, Sergei; Diogenes, Anibal R; Patil, Mayur J; Ginsburg, Erika; Henry, Michael A; Akopian, Armen N

2013-11-29

Prolactin (PRL) regulates activity of nociceptors and causes hyperalgesia in pain conditions. PRL enhances nociceptive responses by rapidly modulating channels in nociceptors. The molecular mechanisms underlying PRL-induced transient signaling in neurons are not well understood. Here we use a variety of cell biology and pharmacological approaches to show that PRL transiently enhanced capsaicin-evoked responses involve protein kinase C ε (PKCε) or phosphatidylinositol 3-kinase (PI3K) pathways in female rat trigeminal (TG) neurons. We next reconstituted PRL-induced signaling in a heterologous expression system and TG neurons from PRL receptor (PRLR)-null mutant mice by expressing rat PRLR-long isoform (PRLR-L), PRLR-short isoform (PRLR-S), or a mix of both. Results show that PRLR-S, but not PRLR-L, is capable of mediating PRL-induced transient enhancement of capsaicin responses in both male and female TG neurons. However, co-expression of PRLR-L with PRLR-S (1:1 ratio) leads to the inhibition of the transient PRL actions. Co-expression of PRLR-L deletion mutants with PRLR-S indicated that the cytoplasmic site adjacent to the trans-membrane domain of PRLR-L was responsible for inhibitory effects of PRLR-L. Furthermore, in situ hybridization and immunohistochemistry data indicate that in normal conditions, PRLR-L is expressed mainly in glia with little expression in rat sensory neurons (3-5%) and human nerves. The predominant PRLR form in TG neurons/nerves from rats and humans is PRLR-S. Altogether, PRL-induced transient signaling in sensory neurons is governed by PI3K or PKCε, mediated via the PRLR-S isoform, and transient effects mediated by PRLR-S are inhibited by presence of PRLR-L in these cells.

The conserved role of Krox-20 in directing Hox gene expression during vertebrate hindbrain segmentation.

PubMed

Nonchev, S; Maconochie, M; Vesque, C; Aparicio, S; Ariza-McNaughton, L; Manzanares, M; Maruthainar, K; Kuroiwa, A; Brenner, S; Charnay, P; Krumlauf, R

1996-09-03

Transient segmentation in the hindbrain is a fundamental morphogenetic phenomenon in the vertebrate embryo, and the restricted expression of subsets of Hox genes in the developing rhombomeric units and their derivatives is linked with regional specification. Here we show that patterning of the vertebrate hindbrain involves the direct upregulation of the chicken and pufferfish group 2 paralogous genes, Hoxb-2 and Hoxa-2, in rhombomeres 3 and 5 (r3 and r5) by the zinc finger gene Krox-20. We identified evolutionarily conserved r3/r5 enhancers that contain high affinity Krox-20. binding sites capable of mediating transactivation by Krox-20. In addition to conservation of binding sites critical for Krox-20 activity in the chicken Hoxa-2 and pufferfish Hoxb-2 genes, the r3/r5 enhancers are also characterized by the presence of a number of identical motifs likely to be involved in cooperative interactions with Krox-20 during the process of hindbrain patterning in vertebrates.

Transient Gene Expression in Maize, Rice, and Wheat Cells Using an Airgun Apparatus 1

PubMed Central

Oard, James H.; Paige, David F.; Simmonds, John A.; Gradziel, Thomas M.

1990-01-01

An airgun apparatus has been constructed for transient gene expression studies of monocots. This device utilizes compressed air from a commercial airgun to propel macroprojectile and DNA-coated tungsten particles. The β-glucuronidase (GUS) reporter gene was used to monitor transient expression in three distinct cell types of maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum). The highest level of GUS activity in cultured maize cells was observed when distance between stopping plate and target cells was adjusted to 4.3 centimeters. Efficiency of transformation was estimated to be 4.4 × 10−3. In a partial vacuum of 700 millimeters Hg, velocity of macroprojectile was measured at 520 meters per second with a 6% reduction in velocity at atmospheric pressure. A polyethylene film placed in the breech before firing contributed to a 12% increase in muzzle velocity. A 700 millimeters Hg level of vacuum was necessary for maximum number of transfornants. GUS expression was also detected in wheat leaf base tissue of microdissected shoot apices. High levels of transient gene expression were also observed in hard, compact embryogenic callus of rice. These results show that the airgun apparatus is a convenient, safe, and low-cost device for rapid transient gene expression studies in cereals. Images Figure 7 Figure 8 Figure 9 PMID:16667278

Binocular pattern deprivation interferes with the expression of proteins involved in primary visual cortex maturation in the cat.

PubMed

Laskowska-Macios, Karolina; Nys, Julie; Hu, Tjing-Tjing; Zapasnik, Monika; Van der Perren, Anke; Kossut, Malgorzata; Burnat, Kalina; Arckens, Lutgarde

2015-08-14

Binocular pattern deprivation from eye opening (early BD) delays the maturation of the primary visual cortex. This delay is more pronounced for the peripheral than the central visual field representation within area 17, particularly between the age of 2 and 4 months [Laskowska-Macios, Cereb Cortex, 2014]. In this study, we probed for related dynamic changes in the cortical proteome. We introduced age, cortical region and BD as principal variables in a 2-D DIGE screen of area 17. In this way we explored the potential of BD-related protein expression changes between central and peripheral area 17 of 2- and 4-month-old BD (2BD, 4BD) kittens as a valid parameter towards the identification of brain maturation-related molecular processes. Consistent with the maturation delay, distinct developmental protein expression changes observed for normal kittens were postponed by BD, especially in the peripheral region. These BD-induced proteomic changes suggest a negative regulation of neurite outgrowth, synaptic transmission and clathrin-mediated endocytosis, thereby implicating these processes in normal experience-induced visual cortex maturation. Verification of the expression of proteins from each of the biological processes via Western analysis disclosed that some of the transient proteomic changes correlate to the distinct behavioral outcome in adult life, depending on timing and duration of the BD period [Neuroscience 2013;255:99-109]. Taken together, the plasticity potential to recover from BD, in relation to ensuing restoration of normal visual input, appears to rely on specific protein expression changes and cellular processes induced by the loss of pattern vision in early life.

ramosa2 encodes a LATERAL ORGAN BOUNDARY domain protein that determines the fate of stem cells in branch meristems of maize.

PubMed

Bortiri, Esteban; Chuck, George; Vollbrecht, Erik; Rocheford, Torbert; Martienssen, Rob; Hake, Sarah

2006-03-01

Genetic control of grass inflorescence architecture is critical given that cereal seeds provide most of the world's food. Seeds are borne on axillary branches, which arise from groups of stem cells in axils of leaves and whose branching patterns dictate most of the variation in plant form. Normal maize (Zea mays) ears are unbranched, and tassels have long branches only at their base. The ramosa2 (ra2) mutant of maize has increased branching with short branches replaced by long, indeterminate ones. ra2 was cloned by chromosome walking and shown to encode a LATERAL ORGAN BOUNDARY domain transcription factor. ra2 is transiently expressed in a group of cells that predicts the position of axillary meristem formation in inflorescences. Expression in different mutant backgrounds places ra2 upstream of other genes that regulate branch formation. The early expression of ra2 suggests that it functions in the patterning of stem cells in axillary meristems. Alignment of ra2-like sequences reveals a grass-specific domain in the C terminus that is not found in Arabidopsis thaliana. The ra2-dm allele suggests this domain is required for transcriptional activation of ra1. The ra2 expression pattern is conserved in rice (Oryza sativa), barley (Hordeum vulgare), sorghum (Sorghum bicolor), and maize, suggesting that ra2 is critical for shaping the initial steps of grass inflorescence architecture.

Segmental heterogeneity in Bcl-2, Bcl-xL and Bax expression in rat tubular epithelium after ischemia-reperfusion.

PubMed

Valdés, Francisco; Pásaro, Eduardo; Díaz, Inmaculada; Centeno, Alberto; López, Eduardo; García-Doval, Sandra; González-Roces, Severino; Alba, Alfonso; Laffon, Blanca

2008-06-01

Studies in rats with bilateral clamping of renal arteries showed transient Bcl-2, Bcl-xL and Bax expression in renal tubular epithelium following ischemia-reperfusion. However, current data on the preferential localization of specific mRNAs or proteins are limited because gene expression was not analysed at segmental level. This study analyses the mRNA expression of Bcl-2, Bcl-xL and Bax in four segments of proximal and distal tubules localized in the renal cortex and outer medulla in rat kidneys with bilateral renal clamping for 30 min and seven reperfusion times versus control animals without clamp. Proximal convoluted tubule (PCT), distal convoluted tubule (DCT), proximal straight tubule (PST) and medullary thick ascending limb (MTAL) were obtained by manual microdissection. RT-PCR was used to analyse mRNA expression at segmental level. Proximal convoluted tubule and MTAL showed early, persistent and balanced up-regulation of Bcl-2, Bcl-xL and Bax, while PST and DCT revealed only Bcl-2 and Bcl-xL, when only Bax was detected in PST. DCT expressed Bcl-xL initially, and persistent Bcl-2 later. These patterns suggest a heterogeneous apoptosis regulatory response in rat renal tubules after ischemia-reperfusion, independently of cortical or medullary location. This heterogeneity of the expression patterns of Bcl-2 genes could explain the different susceptibility to undergo apoptosis, the different threshold to ischemic damage and the different adaptive capacity to injury among these tubular segments.

Imaging Neural Activity Using Thy1-GCaMP Transgenic mice

PubMed Central

Chen, Qian; Cichon, Joseph; Wang, Wenting; Qiu, Li; Lee, Seok-Jin R.; Campbell, Nolan R.; DeStefino, Nicholas; Goard, Michael J.; Fu, Zhanyan; Yasuda, Ryohei; Looger, Loren L.; Arenkiel, Benjamin R.; Gan, Wen-Biao; Feng, Guoping

2014-01-01

Summary The ability to chronically monitor neuronal activity in the living brain is essential for understanding the organization and function of the nervous system. The genetically encoded green fluorescent protein based calcium sensor GCaMP provides a powerful tool for detecting calcium transients in neuronal somata, processes, and synapses that are triggered by neuronal activities. Here we report the generation and characterization of transgenic mice that express improved GCaMPs in various neuronal subpopulations under the control of the Thy1 promoter. In vitro and in vivo studies show that calcium transients induced by spontaneous and stimulus-evoked neuronal activities can be readily detected at the level of individual cells and synapses in acute brain slices, as well as chronically in awake behaving animals. These GCaMP transgenic mice allow investigation of activity patterns in defined neuronal populations in the living brain, and will greatly facilitate dissecting complex structural and functional relationships of neural networks. PMID:23083733

Particle bombardment - mediated gene transfer and GFP transient expression in Seteria viridis.

PubMed

Mookkan, Muruganantham

2018-04-03

Setaria viridis is one of the most important model grasses in studying monocot plant biology. Transient gene expression study is a very important tool in plant biotechnology, functional genomics, and CRISPR-Cas9 genome editing technology via particle bombardment. In this study, a particle bombardment-mediated protocol was developed to introduce DNA into Setaria viridis in vitro leaf explants. In addition, physical and biological parameters, such as helium pressure, distance from stopping screen to the target tissues, DNA concentration, and number of bombardments, were tested and optimized. Optimum concentration of transient GFP expression was achieved using 1.5 ug plasmid DNA with 0.6 mm gold particles and 6 cm bombardment distance, using 1,100 psi. Doubling the bombardment instances provides the maximum number of foci of transient GFP expression. This simple protocol will be helpful for genomics studies in the S. viridis monocot model.

Exposure of mice to secondhand smoke elicits both transient and long-lasting transcriptional changes in cancer-related functional networks.

PubMed

Tommasi, Stella; Zheng, Albert; Besaratinia, Ahmad

2015-05-15

Secondhand smoke (SHS) has long been linked to lung cancer and other diseases in nonsmokers. Yet, the underlying mechanisms of SHS carcinogenicity in nonsmokers remain to be elucidated. We investigated the immediate and long-lasting effects of SHS exposure on gene expression in mice in vivo. We exposed mice whole body to SHS for 5 h/day, 5 days/week for 4 months in exposure chambers of a microprocessor-controlled smoking machine. Subsequently, we performed microarray gene expression profiling, genome-wide, to construct the pulmonary transcriptome of SHS-exposed mice, immediately after discontinuation of exposure (T0) and following 1-month (T1) and 7-month (T2) recoveries in clean air. Sub-chronic exposure of mice to SHS elicited a robust transcriptomic response, including both reversible and irreversible changes in gene expression. There were 674 differentially expressed transcripts immediately after treatment (T0), of which the majority were involved in xenobiotic metabolism, signaling, and innate immune response. Reduced, yet, substantial numbers of differentially expressed transcripts were detectable in mice after cessation of SHS-exposure (254 transcripts at T1 and 30 transcripts at T2). Top biofunctional networks disrupted in SHS-exposed mice, even after termination of exposure, were implicated in cancer, respiratory disease, and inflammatory disease. Our data show that exposure of mice to SHS induces both transient and long-lasting changes in gene expression, which impact cancer-related functional networks. The pattern of transcriptional changes in SHS-exposed mice may provide clues on the underlying mechanisms of lung tumorigenesis in nonsmokers. Our findings underscore the importance of eliminating SHS from environments where nonsmokers are unavoidably exposed to this carcinogen. © 2014 UICC.

Heat sensitization in skin and muscle nociceptors expressing distinct combinations of TRPV1 and TRPV2 protein.

PubMed

Rau, K K; Jiang, N; Johnson, R D; Cooper, B Y

2007-04-01

Recordings were made from small and medium diameter dorsal root ganglia (DRG) neurons that expressed transient receptor potential (TRP) proteins. Physiologically characterized skin nociceptors expressed either TRPV1 (type 2) or TRPV2 (type 4) in isolation. Other nociceptors co-expressed both TRP proteins and innervated deep tissue sites (gastrocnemius muscle, distal colon; type 5, type 8) and skin (type 8). Subpopulations of myelinated (type 8) and unmyelinated (type 5) nociceptors co-expressed both TRPs. Cells that expressed TRPV1 were excellent transducers of intense heat. Proportional inward currents were obtained from a threshold of approximately 46.5 to approximately 56 degrees C. In contrast, cells expressing TRPV2 alone (52 degrees C threshold) did not reliably transduce the intensity of thermal events. Studies were undertaken to assess the capacity of skin and deep nociceptors to exhibit sensitization to repeated intense thermal stimuli [heat-heat sensitization (HHS)]. Only nociceptors that expressed TRPV2, alone or in combination with TRPV1, exhibited HHS. HHS was shown to be Ca(2+) dependent in either case. Intracellular Ca(2+) dependent pathways to HHS varied with the pattern of TRP protein expression. Cells co-expressing both TRPs modulated heat reactivity through serine/threonine phosphorylation or PLA(2)-dependent pathways. Cells expressing only TRPV2 may have relied on tyrosine kinases for HHS. We conclude that heat sensitization in deep and superficial capsaicin and capsaicin-insensitive C and Adelta nociceptors varies with the distribution of TRPV1 and TRPV2 proteins. The expression pattern of these proteins are specific to subclasses of physiologically identified C and A fiber nociceptors with highly restricted tissue targets.

Self-reflection and positive schizotypy in the adolescent brain.

PubMed

Debbané, Martin; Vrtička, Pascal; Lazouret, Marine; Badoud, Deborah; Sander, David; Eliez, Stephan

2014-01-01

Clinical and phenomenological accounts of schizophrenia suggest that impairments in self-reflective processes significantly contribute to psychopathological expression. Recent imaging studies observe atypical cerebral activation patterns during self-reflection, especially around the cortical midline structures, both in psychosis-prone adults and individuals with schizophrenia. Given that self-reflection processes consolidate during adolescence, and that early transient expression of psychosis (positive schizotypy) also arises during this period, the present study sought to examine whether atypical cerebral activation during self-reflection task could be associated with early schizotypic expression during adolescence. Forty-two neurotypical adolescent participants (19 females) aged from 12 to 19 (15.92±1.9) underwent a self-reflection task using functional neuroimaging (fMRI), where they had to evaluate trait adjectives (1 to 4 ratings) about themselves or their same sex best friend. The Schizotypal Personality Questionnaire (SPQ) was employed to assess positive schizotypic expression. Results showed that positive schizotypy in adolescents significantly correlated with cortical midline activation patterns in the dorsomedial prefrontal cortex (dmPFC) and the posterior cingulate cortex (PCC), as well as the dorsolateral PFC and the lingual gyrus. The results are consistent with previous imaging literature on self-reflection and schizophrenia. They further highlight that the relationship between self-reflection processes and positive schizotypy operates at the trait level of expression and can be observed as early as adolescence. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of self-coupling and asymmetric output on metastable dynamical transient firing patterns in arrays of neurons with bidirectional inhibitory coupling.

PubMed

Horikawa, Yo

2016-04-01

Metastable dynamical transient patterns in arrays of bidirectionally coupled neurons with self-coupling and asymmetric output were studied. First, an array of asymmetric sigmoidal neurons with symmetric inhibitory bidirectional coupling and self-coupling was considered and the bifurcations of its steady solutions were shown. Metastable dynamical transient spatially nonuniform states existed in the presence of a pair of spatially symmetric stable solutions as well as unstable spatially nonuniform solutions in a restricted range of the output gain of a neuron. The duration of the transients increased exponentially with the number of neurons up to the maximum number at which the spatially nonuniform steady solutions were stabilized. The range of the output gain for which they existed reduced as asymmetry in a sigmoidal output function of a neuron increased, while the existence range expanded as the strength of inhibitory self-coupling increased. Next, arrays of spiking neuron models with slow synaptic inhibitory bidirectional coupling and self-coupling were considered with computer simulation. In an array of Class 1 Hindmarsh-Rose type models, in which each neuron showed a graded firing rate, metastable dynamical transient firing patterns were observed in the presence of inhibitory self-coupling. This agreed with the condition for the existence of metastable dynamical transients in an array of sigmoidal neurons. In an array of Class 2 Bonhoeffer-van der Pol models, in which each neuron had a clear threshold between firing and resting, long-lasting transient firing patterns with bursting and irregular motion were observed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cell stress and translational inhibitors transiently increase the abundance of mammalian SINE transcripts.

PubMed Central

Liu, W M; Chu, W M; Choudary, P V; Schmid, C W

1995-01-01

The abundance of Alu RNA is transiently increased by heat shock in human cell lines. This effect is specific to Alu repeats among Pol III transcribed genes, since the abundance of 7SL, 7SK, 5S and U6 RNAs is essentially unaffected by heat shock. The rapid induction of Alu expression precedes the heat shock induction of mRNAs for the ubiquitin and HSP 70 heat shock genes. Heat shock mimetics also transiently induce Alu expression indicating that increased Alu expression is a general cell-stress response. Cycloheximide treatment rapidly and transiently increases the abundance of Alu RNA. Again, compared with other genes transcribed by Pol III, this increase is specific to Alu. However, as distinguished from the cell stress response, cycloheximide does not induce expression of HSP 70 and ubiquitin mRNAs. Puromycin also increases Alu expression, suggesting that this response is generally caused by translational inhibition. The response of mammalian SINEs to cell stress and translational inhibition is not limited to SINEs which are Alu homologues. Heat shock and cycloheximide each transiently induce Pol III directed expression of B1 and B2 RNAs in mouse cells and C-element RNA in rabbit cells. Together, these three species exemplify the known SINE composition of placental mammals, suggesting that mammalian SINEs are similarly regulated and may serve a common function. Images PMID:7784180

An Ankyrin Repeat-Containing Protein, Characterized as a Ubiquitin Ligase, Is Closely Associated with Membrane-Enclosed Organelles and Required for Pollen Germination and Pollen Tube Growth in Lily1[W

PubMed Central

Huang, Jian; Chen, Feng; Del Casino, Cecilia; Autino, Antonella; Shen, Mouhua; Yuan, Shuai; Peng, Jia; Shi, Hexin; Wang, Chen; Cresti, Mauro; Li, Yiqin

2006-01-01

Exhibiting rapid polarized growth, the pollen tube delivers the male gametes into the ovule for fertilization in higher plants. To get an overall picture of gene expression during pollen germination and pollen tube growth, we profiled the transcription patterns of 1,536 pollen cDNAs from lily (Lilium longiflorum) by microarray. Among those that exhibited significant differential expression, a cDNA named lily ankyrin repeat-containing protein (LlANK) was thoroughly studied. The full-length LlANK cDNA sequence predicts a protein containing five tandem ankyrin repeats and a RING zinc-finger domain. The LlANK protein possesses ubiquitin ligase activity in vitro. RNA blots demonstrated that LlANK transcript is present in mature pollen and its level, interestingly contrary to most pollen mRNAs, up-regulated significantly during pollen germination and pollen tube growth. When fused with green fluorescent protein and transiently expressed in pollen, LlANK was found dominantly associated with membrane-enclosed organelles as well as the generative cell. Overexpression of LlANK, however, led to abnormal growth of the pollen tube. On the other hand, transient silencing of LlANK impaired pollen germination and tube growth. Taken together, these results showed that LlANK is a ubiquitin ligase associated with membrane-enclosed organelles and required for polarized pollen tube growth. PMID:16461387

Palm is expressed in both developing and adult mouse lens and retina

PubMed Central

Castellini, Meryl; Wolf, Louise V; Chauhan, Bharesh K; Galileo, Deni S; Kilimann, Manfred W; Cvekl, Ales; Duncan, Melinda K

2005-01-01

Background Paralemmin (Palm) is a prenyl-palmitoyl anchored membrane protein that can drive membrane and process formation in neurons. Earlier studies have shown brain preferred Palm expression, although this protein is a major water insoluble protein in chicken lens fiber cells and the Palm gene may be regulated by Pax6. Methods The expression profile of Palm protein in the embryonic, newborn and adult mouse eye as well as dissociated retinal neurons was determined by confocal immunofluorescence. The relative mRNA levels of Palm, Palmdelphin (PalmD) and paralemmin2 (Palm2) in the lens and retina were determined by real time rt-PCR. Results In the lens, Palm is already expressed at 9.5 dpc in the lens placode, and this expression is maintained in the lens vesicle throughout the formation of the adult lens. Palm is largely absent from the optic vesicle but is detectable at 10.5 dpc in the optic cup. In the developing retina, Palm expression transiently upregulates during the formation of optic nerve as well as in the formation of both the inner and outer plexiform layers. In short term dissociated chick retinal cultures, Palm protein is easily detectable, but the levels appear to reduce sharply as the cultures age. Palm mRNA was found at much higher levels relative to Palm2 or PalmD in both the retina and lens. Conclusion Palm is the major paralemmin family member expressed in the retina and lens and its expression in the retina transiently upregulates during active neurite outgrowth. The expression pattern of Palm in the eye is consistent with it being a Pax6 responsive gene. Since Palm is known to be able to drive membrane formation in brain neurons, it is possible that this molecule is crucial for the increase in membrane formation during lens fiber cell differentiation. PMID:15969763

Weather chains during the 2013/2014 winter and their significance for seasonal prediction

NASA Astrophysics Data System (ADS)

Davies, Huw C.

2015-11-01

Day-to-day weather forecasting has improved substantially over the past few decades. In contrast, progress in seasonal prediction outside the tropics has been meagre and mixed. On seasonal timescales, the constraining influence of the initial atmospheric state is weak, and the internal variability associated with transient weather systems tends to be large compared with the nuanced influence of anomalies in external forcing. Current research and operational activities focus on exploring and exploiting potential links between external anomalies and seasonal-mean climate patterns. Here I examine reanalysed meteorological data sets for the unusual winter 2013/2014, with drought and freezing conditions juxtaposed over North America and severe wet and stormy weather over parts of Europe, to study the role of weather systems and their transient upper-tropospheric flow patterns. I find that the amplitude, recurrence and location of these transient patterns account directly for the corresponding anomalous seasonal-mean patterns. They occurred episodically and sequentially, were linked dynamically, and exhibited some circumpolar connectivity. I conclude that the upper-tropospheric components of transient weather systems are significant for understanding and predicting seasonal weather patterns, whereas the role of external factors is more subtle.

Developmental and cell-specific expression of thyroid hormone transporters in the mouse cochlea.

PubMed

Sharlin, David S; Visser, Theo J; Forrest, Douglas

2011-12-01

Thyroid hormone is essential for the development of the cochlea and auditory function. Cochlear response tissues, which express thyroid hormone receptor β (encoded by Thrb), include the greater epithelial ridge and sensory epithelium residing inside the bony labyrinth. However, these response tissues lack direct blood flow, implying that mechanisms exist to shuttle hormone from the circulation to target tissues. Therefore, we investigated expression of candidate thyroid hormone transporters L-type amino acid transporter 1 (Lat1), monocarboxylate transporter (Mct)8, Mct10, and organic anion transporting polypeptide 1c1 (Oatp1c1) in mouse cochlear development by in situ hybridization and immunofluorescence analysis. L-type amino acid transporter 1 localized to cochlear blood vessels and transiently to sensory hair cells. Mct8 localized to the greater epithelial ridge, tympanic border cells underlying the sensory epithelium, spiral ligament fibrocytes, and spiral ganglion neurons, partly overlapping with the Thrb expression pattern. Mct10 was detected in a highly restricted pattern in the outer sulcus epithelium and weakly in tympanic border cells and hair cells. Organic anion transporting polypeptide 1c1 localized primarily to fibrocytes in vascularized tissues of the spiral limbus and spiral ligament and to tympanic border cells. Investigation of hypothyroid Tshr(-/-) mice showed that transporter expression was delayed consistent with retardation of cochlear tissue maturation but not with compensatory responses to hypothyroidism. The results demonstrate specific expression of thyroid hormone transporters in the cochlea and suggest that a network of thyroid hormone transport underlies cochlear development.

Expression and Functional Analysis of WRKY Transcription Factors in Chinese Wild Hazel, Corylus heterophylla Fisch

PubMed Central

Liang, Li-Song; Ma, Qing-Hua; Chen, Xin; Zong, Jian-Wei; Wang, Gui-Xi

2015-01-01

Plant WRKY transcription factors are known to regulate various biotic and abiotic stress responses. In this study we identified a total of 30 putative WRKY unigenes in a transcriptome dataset of the Chinese wild Hazel, Corylus heterophylla, a species that is noted for its cold tolerance. Thirteen full-length of these ChWRKY genes were cloned and found to encode complete protein sequences, and they were divided into three groups, based on the number of WRKY domains and the pattern of zinc finger structures. Representatives of each of the groups, Unigene25835 (group I), Unigene37641 (group II) and Unigene20441 (group III), were transiently expressed as fusion proteins with yellow fluorescent fusion protein in Nicotiana benthamiana, where they were observed to accumulate in the nucleus, in accordance with their predicted roles as transcriptional activators. An analysis of the expression patterns of all 30 WRKY genes revealed differences in transcript abundance profiles following exposure to cold, drought and high salinity conditions. Among the stress-inducible genes, 23 were up-regulated by all three abiotic stresses and the WRKY genes collectively exhibited four different patterns of expression in flower buds during the overwintering period from November to April. The organ/tissue related expression analysis showed that 18 WRKY genes were highly expressed in stem but only 2 (Unigene9262 and Unigene43101) were greatest in male anthotaxies. The expression of Unigene37641, a member of the group II WRKY genes, was substantially up-regulated by cold, drought and salinity treatments, and its overexpression in Arabidopsis thaliana resulted in better seedling growth, compared with wild type plants, under cold treatment conditions. The transgenic lines also had exhibited higher soluble protein content, superoxide dismutase and peroxidase activiety and lower levels of malondialdehyde, which collectively suggets that Unigene37641 expression promotes cold tolerance. PMID:26270529

Expression and Functional Analysis of WRKY Transcription Factors in Chinese Wild Hazel, Corylus heterophylla Fisch.

PubMed

Zhao, Tian-Tian; Zhang, Jin; Liang, Li-Song; Ma, Qing-Hua; Chen, Xin; Zong, Jian-Wei; Wang, Gui-Xi

2015-01-01

Plant WRKY transcription factors are known to regulate various biotic and abiotic stress responses. In this study we identified a total of 30 putative WRKY unigenes in a transcriptome dataset of the Chinese wild Hazel, Corylus heterophylla, a species that is noted for its cold tolerance. Thirteen full-length of these ChWRKY genes were cloned and found to encode complete protein sequences, and they were divided into three groups, based on the number of WRKY domains and the pattern of zinc finger structures. Representatives of each of the groups, Unigene25835 (group I), Unigene37641 (group II) and Unigene20441 (group III), were transiently expressed as fusion proteins with yellow fluorescent fusion protein in Nicotiana benthamiana, where they were observed to accumulate in the nucleus, in accordance with their predicted roles as transcriptional activators. An analysis of the expression patterns of all 30 WRKY genes revealed differences in transcript abundance profiles following exposure to cold, drought and high salinity conditions. Among the stress-inducible genes, 23 were up-regulated by all three abiotic stresses and the WRKY genes collectively exhibited four different patterns of expression in flower buds during the overwintering period from November to April. The organ/tissue related expression analysis showed that 18 WRKY genes were highly expressed in stem but only 2 (Unigene9262 and Unigene43101) were greatest in male anthotaxies. The expression of Unigene37641, a member of the group II WRKY genes, was substantially up-regulated by cold, drought and salinity treatments, and its overexpression in Arabidopsis thaliana resulted in better seedling growth, compared with wild type plants, under cold treatment conditions. The transgenic lines also had exhibited higher soluble protein content, superoxide dismutase and peroxidase activiety and lower levels of malondialdehyde, which collectively suggets that Unigene37641 expression promotes cold tolerance.

Screening promoters for Anthurium transformation using transient expression

USDA-ARS?s Scientific Manuscript database

Different promoters and tissue types were evaluated for transient '-glucoronidase (GUS) expression in Anthurium andreanum Hort. ‘Marian Seefurth’ following microprojectile bombardment. Plasmids containing the Ubiquitin 2, Actin 1, Cytochrome C1 from rice, Ubiquitin 1 from maize and 35 S promoter fr...

Transient Expression of Chimeric Fluorescent Reporter Proteins in Pollen Tubes to Study Protein Polar Secretion and Dynamics.

PubMed

Zhong, Guitao; Liu, Ronghe; Zhuang, Menglong; Wang, Hao

2017-01-01

Transient expression of chimeric fluorescent reporter proteins by biolistic bombardment is a quick and useful procedure for studying subcellular protein localization and dynamics in plants. It is especially beneficial in specific plant cells which are not suitable for protoplast-based and Agrobacterium-mediated protein transient expression. Polar protein secretion and vesicular trafficking play essential functions for cell polarization and tip growth. The growing pollen tube is regarded as an ideal model plant cell system to study the machinery and regulation of polar protein trafficking and targeting. A large amount of newly synthesized proteins are packed and polarly transported to the apical region to support the rapid and highly polarized tip growth. Here, we described a detailed step-by-step protocol for the transient expression of chimeric fluorescent reporter proteins in growing Arabidopsis and tobacco pollen tubes to study polar transportation logistics and mechanisms. In addition, we have optimized the Arabidopsis and tobacco in vitro pollen germination medium and the conditions to maximize the efficiency of protein expression. As a proof of concept, we have used this protocol to express actin microfilament and late endosomal fluorescent markers in Arabidopsis and tobacco pollen tubes.

And yet it moves! Involving transient flow conditions is the logical next step for WHPA analysis

NASA Astrophysics Data System (ADS)

Rodriguez-Pretelin, A.; Nowak, W.

2017-12-01

As the first line of defense among different safety measures, Wellhead Protection Areas (WHPAs) have been broadly used to protect drinking water wells against sources of pollution. In most cases, their implementation relies on simplifications, such as assuming homogeneous or zonated aquifer conditions or considering steady-state flow scenarios. Obviously, both assumptions inevitably invoke errors. However, while uncertainty due to aquifer heterogeneity has been extensively studied in the literature, the impact of transient flow conditions have received yet very little attention. For instance, WHPA maps in the offices of water supply companies are fixed maps derived from steady-state models although the actual catchment out there are transient. To mitigate high computational costs, we approximate transiency by means of a dynamic superposition of steady-state flow solutions. Then, we analyze four transient drivers that often appear on the seasonal scale: (I) regional groundwater flow direction, (II) strength of the regional hydraulic gradient, (III) natural recharge to the groundwater and (IV) pumping rate. The integration of transiency in WHPA analysis leads to time-frequency maps. They express for each location the temporal frequency of catchment membership. Furthermore, we account for the uncertainty due to incomplete knowledge on geological and transiency conditions, solved through Monte Carlo simulations. The main contribution of this study, is to show the need of enhancing groundwater well protection by considering transient flow considerations during WHPA analysis. To support and complement our statement, we demonstrate that 1) each transient driver imprints an individual spatial pattern in the required WHPA, ranking their influence through a global sensitivity analysis. 2) We compare the influence of transient conditions compared to geological uncertainty in terms of areal WHPA demand. 3) We show that considering geological uncertainty alone is insufficient in the presence of transient conditions. 4) We propose a practical decision rule for selecting a proper reliability level protection in the presence of both transiency and geological uncertainty.

Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells

NASA Technical Reports Server (NTRS)

Allen, G. J.; Kwak, J. M.; Chu, S. P.; Llopis, J.; Tsien, R. Y.; Harper, J. F.; Schroeder, J. I.; Evans, M. L. (Principal Investigator)

1999-01-01

Cytoplasmic free calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in plant cells and multiple signal transduction pathways regulate [Ca2+]cyt in stomatal guard cells. Measuring [Ca2+]cyt in guard cells has previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these problems, we have constitutively expressed the pH-independent, green fluorescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al. 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time-dependent measurements of [Ca2+]cyt in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive [Ca2+]cyt transients in guard cells. [Ca2+]cyt changes could be semi-quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca2+]cyt transients with peak values of up to approximately 1.5 microM, whereas ABA-induced [Ca2+]cyt transients had peak values up to approximately 0.6 microM. These values are similar to stimulus-induced [Ca2+]cyt changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCl concentrations, spontaneous calcium transients were observed. As yellow cameleon 2.1 was expressed in all guard cells, [Ca2+]cyt was measured independently in the two guard cells of single stomates for the first time. ABA-induced, calcium-induced or spontaneous [Ca2+]cyt increases were not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP-based cameleon calcium indicators are suitable to measure [Ca2+]cyt changes in guard cells and enable the pattern of [Ca2+]cyt dynamics to be measured with a high level of reproducibility in Arabidopsis cells. This technical advance in combination with cell biological and molecular genetic approaches will become an invaluable tool in the dissection of plant cell signal transduction pathways.

Transient increase in the levels of gamma-tubulin complex in reorientation of cortical microtubules by gravity in azuki bean epicotyls

NASA Astrophysics Data System (ADS)

Soga, Kouichi; Kotake, Toshihisa; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro; Hoson, Takayuki

Azuki bean (Vigna angularis Ohwi et Ohashi) seedlings were exposed to centrifugal hypergravity, and the changes in the orientation of cortical microtubules and the expression of genes cording γ-tubulin complex (VaTUBG and VaSpc98p) were examined. By 300 g treatment, the percentage of cells with transverse microtubules was decreased, while that with longitudinal microtubules was increased in epicotyls. Hypergravity increased the expression of VaTUBG and VaSpc98p transiently. Also, the expression of both genes was increased transiently by removal of hypergravity stimulus. Lanthanum and gadolinium ions, potential blockers of mechanosensitive calcium ion-permeable channels (mechanoreceptors), nullified reorientation of microtubules as well as up-regulation of expression of VaTUBG and VaSpc98p by hypergravity. These results suggest that mechanoreceptors on the plasma membrane may perceive the gravity signal, which leads to reorientation of cortical microtubules by transiently stimulating the formation of γ-tubulin complex.

Automation of large scale transient protein expression in mammalian cells

PubMed Central

Zhao, Yuguang; Bishop, Benjamin; Clay, Jordan E.; Lu, Weixian; Jones, Margaret; Daenke, Susan; Siebold, Christian; Stuart, David I.; Yvonne Jones, E.; Radu Aricescu, A.

2011-01-01

Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI− cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative. PMID:21571074

Transient Inhibition of FGFR2b-Ligands Signaling Leads to Irreversible Loss of Cellular β-Catenin Organization and Signaling in AER during Mouse Limb Development

PubMed Central

Tabatabai, Reza; Baptista, Sheryl; Tiozzo, Caterina; Carraro, Gianni; Wheeler, Matthew; Barreto, Guillermo; Braun, Thomas; Li, Xiaokun; Hajihosseini, Mohammad K.; Bellusci, Saverio

2013-01-01

The vertebrate limbs develop through coordinated series of inductive, growth and patterning events. Fibroblast Growth Factor receptor 2b (FGFR2b) signaling controls the induction of the Apical Ectodermal Ridge (AER) but its putative roles in limb outgrowth and patterning, as well as in AER morphology and cell behavior have remained unclear. We have investigated these roles through graded and reversible expression of soluble dominant-negative FGFR2b molecules at various times during mouse limb development, using a doxycycline/transactivator/tet(O)-responsive system. Transient attenuation (≤24 hours) of FGFR2b-ligands signaling at E8.5, prior to limb bud induction, leads mostly to the loss or truncation of proximal skeletal elements with less severe impact on distal elements. Attenuation from E9.5 onwards, however, has an irreversible effect on the stability of the AER, resulting in a progressive loss of distal limb skeletal elements. The primary consequences of FGFR2b-ligands attenuation is a transient loss of cell adhesion and down-regulation of P63, β1-integrin and E-cadherin, and a permanent loss of cellular β-catenin organization and WNT signaling within the AER. Combined, these effects lead to the progressive transformation of the AER cells from pluristratified to squamous epithelial-like cells within 24 hours of doxycycline administration. These findings show that FGFR2b-ligands signaling has critical stage-specific roles in maintaining the AER during limb development. PMID:24167544

Seasonal Abscisic Acid Signal and a Basic Leucine Zipper Transcription Factor, DkbZIP5, Regulate Proanthocyanidin Biosynthesis in Persimmon Fruit1[C][W][OA

PubMed Central

Akagi, Takashi; Katayama-Ikegami, Ayako; Kobayashi, Shozo; Sato, Akihiko; Kono, Atsushi; Yonemori, Keizo

2012-01-01

Proanthocyanidins (PAs) are secondary metabolites that contribute to plant protection and crop quality. Persimmon (Diospyros kaki) has a unique characteristic of accumulating large amounts of PAs, particularly in its fruit. Normal astringent-type and mutant nonastringent-type fruits show different PA accumulation patterns depending on the seasonal expression patterns of DkMyb4, which is a Myb transcription factor (TF) regulating many PA pathway genes in persimmon. In this study, attempts were made to identify the factors involved in DkMyb4 expression and the resultant PA accumulation in persimmon fruit. Treatment with abscisic acid (ABA) and an ABA biosynthesis inhibitor resulted in differential changes in the expression patterns of DkMyb4 and PA biosynthesis in astringent-type and nonastringent-type fruits depending on the development stage. To obtain an ABA-signaling TF, we isolated a full-length basic leucine zipper (bZIP) TF, DkbZIP5, which is highly expressed in persimmon fruit. We also showed that ectopic DkbZIP5 overexpression in persimmon calluses induced the up-regulation of DkMyb4 and the resultant PA biosynthesis. In addition, a detailed molecular characterization using the electrophoretic mobility shift assay and transient reporter assay indicated that DkbZIP5 recognized ABA-responsive elements in the promoter region of DkMyb4 and acted as a direct regulator of DkMyb4 in an ABA-dependent manner. These results suggest that ABA signals may be involved in PA biosynthesis in persimmon fruit via DkMyb4 activation by DkbZIP5. PMID:22190340

A fluorescent reporter protein containing AtRMR1 domains is targeted to the storage and central vacuoles in Arabidopsis thaliana and tobacco leaf cells.

PubMed

Scabone, Camila María; Frigerio, Lorenzo; Petruccelli, Silvana

2011-10-01

To develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants, the ability of the transmembrane and cytosolic domains of Arabidopsis receptor homology-transmembrane-RING H2-1 (AtRMR1) was evaluated. A secreted version of RFP (secRFP) and a fusion of it to the transmembrane and cytosolic domains of AtRMR1 (RFP-TMCT) were produced and studied both in transient and stable expression assays. Transient expression in leaves of Nicotiana tabacum showed that secRFP is secreted to the apoplast while its fusion to TMCT of AtRMR1 is sufficient to prevent secretion of the reporter. In tobacco leaves, RFP-TMCT reporter showed an endoplasmic reticulum pattern in early expression stages while in late expression stages, it was found in the vacuolar lumen. For the first time, the role of TM and CT domains of AtRMR1 in stable expression in Arabidopsis thaliana is presented; the fusion of TMCT to secRFP is sufficient to sort RFP to the lumen of the central vacuoles in leaves and roots and to the lumen of PSV in cotyledons of mature embryos. In addition, biochemical studies performed in extract from transgenic plants showed that RFP-TMCT is an integral membrane protein. Full-length RFP-TMCT was also found in the vacuolar lumen, suggesting internalization into destination vacuole. Not colocalization of RFP-TMCT with tonoplast and plasma membrane markers were observed. This membrane vacuolar determinant sorting signal could be used for future application in molecular pharming as an alternative means to sort proteins of interest to vacuoles.

Transient chondrogenic phase in the intramembranous pathway during normal skeletal development.

PubMed

Nah, H D; Pacifici, M; Gerstenfeld, L C; Adams, S L; Kirsch, T

2000-03-01

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments.

Sex differences in mouse Transient Receptor Potential Cation Channel, Subfamily M, Member 8 expressing trigeminal ganglion neurons

PubMed Central

Caudle, Stephanie L.; Jenkins, Alan C.; Ahn, Andrew H.; Neubert, John K.

2017-01-01

The detection of cool temperatures is thought to be mediated by primary afferent neurons that express the cool temperature sensing protein Transient Receptor Potential Cation Channel, Subfamily M, Member 8 (TRPM8). Using mice, this study tested the hypothesis that sex differences in sensitivity to cool temperatures were mediated by differences in neurons that express TRPM8. Ion currents from TRPM8 expressing trigeminal ganglion (TRG) neurons in females demonstrated larger hyperpolarization-activated cyclic nucleotide-gated currents (Ih) than male neurons at both 30° and 18°C. Additionally, female neurons’ voltage gated potassium currents (Ik) were suppressed by cooling, whereas male Ik was not significantly affected. At the holding potential tested (-60mV) TRPM8 currents were not visibly activated in either sex by cooling. Modeling the effect of Ih and Ik on membrane potentials demonstrated that at 30° the membrane potential in both sexes is unstable. At 18°, female TRPM8 TRG neurons develop a large oscillating pattern in their membrane potential, whereas male neurons become highly stable. These findings suggest that the differences in Ih and Ik in the TRPM8 TRG neurons of male and female mice likely leads to greater sensitivity of female mice to the cool temperature. This hypothesis was confirmed in an operant reward/conflict assay. Female mice contacted an 18°C surface for approximately half the time that males contacted the cool surface. At 33° and 10°C male and female mice contacted the stimulus for similar amounts of time. These data suggest that sex differences in the functioning of Ih and Ik in TRPM8 expressing primary afferent neurons leads to differences in cool temperature sensitivity. PMID:28472061

Molecular characterization of tocopherol biosynthetic genes in sweetpotato that respond to stress and activate the tocopherol production in tobacco.

PubMed

Ji, Chang Yoon; Kim, Yun-Hee; Kim, Ho Soo; Ke, Qingbo; Kim, Gun-Woo; Park, Sung-Chul; Lee, Haeng-Soon; Jeong, Jae Cheol; Kwak, Sang-Soo

2016-09-01

Tocopherol (vitamin E) is a chloroplast lipid that is presumed to be involved in the plant response to oxidative stress. In this study, we isolated and characterized five tocopherol biosynthetic genes from sweetpotato (Ipomoea batatas [L.] Lam) plants, including genes encoding 4-hydroxyphenylpyruvate dioxygenase (IbHPPD), homogentisate phytyltransferase (IbHPT), 2-methyl-6-phytylbenzoquinol methyltransferase (IbMPBQ MT), tocopherol cyclase (IbTC) and γ-tocopherol methyltransferase (IbTMT). Fluorescence microscope analysis indicated that four proteins localized into the chloroplast, whereas IbHPPD observed in the nuclear. Quantitative RT-PCR analysis revealed that the expression patterns of the five tocopherol biosynthetic genes varied in different plant tissues and under different stress conditions. All five genes were highly expressed in leaf tissues, whereas IbHPPD and IbHPT were highly expressed in the thick roots. The expression patterns of these five genes significantly differed in response to PEG, NaCl and H2O2-mediated oxidative stress. IbHPPD was strongly induced following PEG and H2O2 treatment and IbHPT was strongly induced following PEG treatment, whereas IbMPBQ MT and IbTC were highly expressed following NaCl treatment. Upon infection of the bacterial pathogen Pectobacterium chrysanthemi, the expression of IbHPPD increased sharply in sweetpotato leaves, whereas the expression of the other genes was reduced or unchanged. Additionally, transient expression of the five tocopherol biosynthetic genes in tobacco (Nicotiana bentamiana) leaves resulted in increased transcript levels of the transgenes expressions and tocopherol production. Therefore, our results suggested that the five tocopherol biosynthetic genes of sweetpotato play roles in the stress defense response as transcriptional regulators of the tocopherol production. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz).

PubMed

Wu, Jun-Zheng; Liu, Qin; Geng, Xiao-Shan; Li, Kai-Mian; Luo, Li-Juan; Liu, Jin-Ping

2017-03-14

Cassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced. Fully expanded leaves from in vitro plantlets of Manihot esculenta were used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization. In this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 °C, resulting in the high yield (4.4 × 10 7 protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H + /monosaccharide cotransporter) with our transient expression protocol and a heterologous Arabidopsis transient gene expression system. We optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.

Scale-dependent erosional patterns in steady-state and transient-state landscapes.

PubMed

Tejedor, Alejandro; Singh, Arvind; Zaliapin, Ilya; Densmore, Alexander L; Foufoula-Georgiou, Efi

2017-09-01

Landscape topography is the expression of the dynamic equilibrium between external forcings (for example, climate and tectonics) and the underlying lithology. The magnitude and spatial arrangement of erosional and depositional fluxes dictate the evolution of landforms during both statistical steady state (SS) and transient state (TS) of major landscape reorganization. For SS landscapes, the common expectation is that any point of the landscape has an equal chance to erode below or above the landscape median erosion rate. We show that this is not the case. Afforded by a unique experimental landscape that provided a detailed space-time recording of erosional fluxes and by defining the so-called E50-area curve, we reveal for the first time that there exists a hierarchical pattern of erosion. Specifically, hillslopes and fluvial channels erode more rapidly than the landscape median erosion rate, whereas intervening parts of the landscape in terms of upstream contributing areas (colluvial regime) erode more slowly. We explain this apparent paradox by documenting the dynamic nature of SS landscapes-landscape locations may transition from being a hillslope to being a valley and then to being a fluvial channel due to ridge migration, channel piracy, and small-scale landscape dynamics through time. Under TS conditions caused by increased precipitation, we show that the E50-area curve drastically changes shape during landscape reorganization. Scale-dependent erosional patterns, as observed in this study, suggest benchmarks in evaluating numerical models and interpreting the variability of sampled erosional rates in field landscapes.

Scale-dependent erosional patterns in steady-state and transient-state landscapes

PubMed Central

Tejedor, Alejandro; Singh, Arvind; Zaliapin, Ilya; Densmore, Alexander L.; Foufoula-Georgiou, Efi

2017-01-01

Landscape topography is the expression of the dynamic equilibrium between external forcings (for example, climate and tectonics) and the underlying lithology. The magnitude and spatial arrangement of erosional and depositional fluxes dictate the evolution of landforms during both statistical steady state (SS) and transient state (TS) of major landscape reorganization. For SS landscapes, the common expectation is that any point of the landscape has an equal chance to erode below or above the landscape median erosion rate. We show that this is not the case. Afforded by a unique experimental landscape that provided a detailed space-time recording of erosional fluxes and by defining the so-called E50-area curve, we reveal for the first time that there exists a hierarchical pattern of erosion. Specifically, hillslopes and fluvial channels erode more rapidly than the landscape median erosion rate, whereas intervening parts of the landscape in terms of upstream contributing areas (colluvial regime) erode more slowly. We explain this apparent paradox by documenting the dynamic nature of SS landscapes—landscape locations may transition from being a hillslope to being a valley and then to being a fluvial channel due to ridge migration, channel piracy, and small-scale landscape dynamics through time. Under TS conditions caused by increased precipitation, we show that the E50-area curve drastically changes shape during landscape reorganization. Scale-dependent erosional patterns, as observed in this study, suggest benchmarks in evaluating numerical models and interpreting the variability of sampled erosional rates in field landscapes. PMID:28959728

Transient, Inducible, Placenta-Specific Gene Expression in Mice

PubMed Central

Fan, Xiujun; Petitt, Matthew; Gamboa, Matthew; Huang, Mei; Dhal, Sabita; Druzin, Maurice L.; Wu, Joseph C.

2012-01-01

Molecular understanding of placental functions and pregnancy disorders is limited by the absence of methods for placenta-specific gene manipulation. Although persistent placenta-specific gene expression has been achieved by lentivirus-based gene delivery methods, developmentally and physiologically important placental genes have highly stage-specific functions, requiring controllable, transient expression systems for functional analysis. Here, we describe an inducible, placenta-specific gene expression system that enables high-level, transient transgene expression and monitoring of gene expression by live bioluminescence imaging in mouse placenta at different stages of pregnancy. We used the third generation tetracycline-responsive tranactivator protein Tet-On 3G, with 10- to 100-fold increased sensitivity to doxycycline (Dox) compared with previous versions, enabling unusually sensitive on-off control of gene expression in vivo. Transgenic mice expressing Tet-On 3G were created using a new integrase-based, site-specific approach, yielding high-level transgene expression driven by a ubiquitous promoter. Blastocysts from these mice were transduced with the Tet-On 3G-response element promoter-driving firefly luciferase using lentivirus-mediated placenta-specific gene delivery and transferred into wild-type pseudopregnant recipients for placenta-specific, Dox-inducible gene expression. Systemic Dox administration at various time points during pregnancy led to transient, placenta-specific firefly luciferase expression as early as d 5 of pregnancy in a Dox dose-dependent manner. This system enables, for the first time, reliable pregnancy stage-specific induction of gene expression in the placenta and live monitoring of gene expression during pregnancy. It will be widely applicable to studies of both placental development and pregnancy, and the site-specific Tet-On G3 mouse will be valuable for studies in a broad range of tissues. PMID:23011919

Loss of CDKL5 disrupts respiratory function in mice.

PubMed

Lee, Kun-Ze; Liao, Wenlin

2018-01-01

Cyclin-dependent kinase-like 5 (CDKL5) is an X-linked gene encoding a serine-threonine kinase that is highly expressed in the central nervous system. Mutations in CDKL5 cause neurological and psychiatric symptoms, including early-onset seizures, motor dysfunction, autistic features and sleep breathing abnormalities in patients. It remains to be addressed whether loss of CDKL5 causes respiratory dysfunction in mice. Here, we examined the respiratory pattern of male Cdkl5 -/y mice at 1-3 months of age during resting breathing and respiratory challenge (i.e., hypoxia and hypercapnia) via whole body plethysmography. The results demonstrated that the resting respiratory frequency and tidal volume of Cdkl5 -/y mice was unaltered compared to that of WT mice at 1 month of age. However, these mutant mice exhibit transient reduction in tidal volume during respiratory challenge even the reduction was restored at 2 months of age. Notably, the sigh-breathing pattern was changed in Cdkl5 -/y mice, showing a transient reduction in sigh volume at 1-2 month of age and long-term attenuation of peak expiratory airflow from 1 to 3 month of age. Therefore, loss of CDKL5 causes breathing deficiency, supporting a CDKL5-mediated regulation of respiratory function in mice. Copyright © 2017 Elsevier B.V. All rights reserved.

Inactivity-induced phrenic and hypoglossal motor facilitation are differentially expressed following intermittent vs. sustained neural apnea

PubMed Central

Baertsch, N. A.

2013-01-01

Reduced respiratory neural activity elicits a rebound increase in phrenic and hypoglossal motor output known as inactivity-induced phrenic and hypoglossal motor facilitation (iPMF and iHMF, respectively). We hypothesized that, similar to other forms of respiratory plasticity, iPMF and iHMF are pattern sensitive. Central respiratory neural activity was reversibly reduced in ventilated rats by hyperventilating below the CO2 apneic threshold to create brief intermittent neural apneas (5, ∼1.5 min each, separated by 5 min), a single brief massed neural apnea (7.5 min), or a single prolonged neural apnea (30 min). Upon restoration of respiratory neural activity, long-lasting (>60 min) iPMF was apparent following brief intermittent and prolonged, but not brief massed, neural apnea. Further, brief intermittent and prolonged neural apnea elicited an increase in the maximum phrenic response to high CO2, suggesting that iPMF is associated with an increase in phrenic dynamic range. By contrast, only prolonged neural apnea elicited iHMF, which was transient in duration (<15 min). Intermittent, massed, and prolonged neural apnea all elicited a modest transient facilitation of respiratory frequency. These results indicate that iPMF, but not iHMF, is pattern sensitive, and that the response to respiratory neural inactivity is motor pool specific. PMID:23493368

Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves.

PubMed

Wu, Su-Li; Yang, Xiao-Bing; Liu, Li-Qun; Jiang, Tao; Wu, Hai; Su, Chao; Qian, Yong-Hua; Jiao, Feng

2015-01-01

To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD600 of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.

Results from a model-independent method of monitoring a geodetic network for patterns of transient deformation

NASA Technical Reports Server (NTRS)

Hurst, Kenneth; Granat, Robert

2005-01-01

We have implmented two multi-station detectors for transient crustal deformation within the Southern California Integrated GPS (SCGIN). One the the primary goals of SCIGN is to detect transient deformation associated with the earthquake cycle in Southern California.

Using Rising Limb Analysis to Estimate Uptake of Reactive Solutes in Advective and Transient Storage Sub-compartments of Stream Ecosystems

NASA Astrophysics Data System (ADS)

Thomas, S. A.; Valett, H.; Webster, J. R.; Mulholland, P. J.; Dahm, C. N.

2001-12-01

Identifying the locations and controls governing solute uptake is a recent area of focus in studies of stream biogeochemistry. We introduce a technique, rising limb analysis (RLA), to estimate areal nitrate uptake in the advective and transient storage (TS) zones of streams. RLA is an inverse approach that combines nutrient spiraling and transient storage modeling to calculate total uptake of reactive solutes and the fraction of uptake occurring within the advective sub-compartment of streams. The contribution of the transient storage zones to solute loss is determined by difference. Twelve-hour coinjections of conservative (Cl-) and reactive (15NO3) tracers were conducted seasonally in several headwater streams among which AS/A ranged from 0.01 - 2.0. TS characteristics were determined using an advection-dispersion model modified to include hydrologic exchange with a transient storage compartment. Whole-system uptake was determined by fitting the longitudinal pattern of NO3 to first-order, exponential decay model. Uptake in the advective sub-compartment was determined by collecting a temporal sequence of samples from a single location beginning with the arrival of the solute front and concluding with the onset of plateau conditions (i.e. the rising limb). Across the rising limb, 15NO3:Cl was regressed against the percentage of water that had resided in the transient storage zone (calculated from the TS modeling). The y-intercept thus provides an estimate of the plateau 15NO3:Cl ratio in the absence of NO3 uptake within the transient storage zone. Algebraic expressions were used to calculate the percentage of NO3 uptake occurring in the advective and transient storage sub-compartments. Application of RLA successfully estimated uptake coefficients for NO3 in the subsurface when the physical dimensions of that habitat were substantial (AS/A > 0.2) and when plateau conditions at the sampling location consisted of waters in which at least 25% had resided in the transient storage zone. In those cases, the TS zone accounted for 8 - 47 % of overall NO3 uptake and uptake rates within the subsurface ranged from 0.7 - 14.3 mg N m-2 d-1.

AFLP-based transcript profiling for cassava genome-wide expression analysis in the onset of storage root formation.

PubMed

Sojikul, Punchapat; Kongsawadworakul, Panida; Viboonjun, Unchera; Thaiprasit, Jittrawan; Intawong, Burapat; Narangajavana, Jarunya; Svasti, Mom Rajawong Jisnuson

2010-10-01

Cassava (Manihot esculenta Crantz) is a root crop that accumulates large quantities of starch, and it is an important source of carbohydrate. Study on gene expressions during storage root development provides important information on storage root formation and starch accumulation as well as unlock new traits for improving of starch yield. cDNA-Amplified Fragment Length Polymorphism (AFLP) was used to compare gene expression profiles in fibrous and storage roots of cassava cultivar Kasetsart 50. Total of 155 differentially expressed transcript-derived fragments with undetectable or low expression in leaves were characterized and classified into 11 groups regarding to their functions. The four major groups were no similarity (20%), hypothetical or unknown proteins (17%), cellular metabolism and biosynthesis (17%) and cellular communication and signaling (14%). Interestingly, sulfite reductase (MeKD82), calcium-dependent protein kinase (CDPK) (MeKD83), ent-kaurene synthase (KS) (MeKD106) and hexose transporter (HT) (MeKD154) showed root-specific expression patterns. This finding is consistent with previously reported genes involved in the initiation of potato tuber. Semi-quantitative reverse transcription polymerase chain reaction of early-developed root samples confirmed that those four genes exhibited significant expression with similar pattern in the storage root initiation and early developmental stages. We proposed that KS and HT may involve in transient induction of CDPK expression, which may play an important role in the signaling pathway of storage root initiation. Sulfite reductase, on the other hand, may involve in storage root development by facilitating sulfur-containing protein biosynthesis or detoxifying the cyanogenic glucoside content through aspartate biosynthesis. Copyright © Physiologia Plantarum 2010.

Dipeptidyl peptidase IV, aminopeptidase N and DPIV/APN-like proteases in cerebral ischemia

PubMed Central

2012-01-01

Background Cerebral inflammation is a hallmark of neuronal degeneration. Dipeptidyl peptidase IV, aminopeptidase N as well as the dipeptidyl peptidases II, 8 and 9 and cytosolic alanyl-aminopeptidase are involved in the regulation of autoimmunity and inflammation. We studied the expression, localisation and activity patterns of these proteases after endothelin-induced occlusion of the middle cerebral artery in rats, a model of transient and unilateral cerebral ischemia. Methods Male Sprague-Dawley rats were used. RT-PCR, immunohistochemistry and protease activity assays were performed at different time points, lasting from 2 h to 7 days after cerebral ischemia. The effect of protease inhibitors on ischemia-dependent infarct volumes was quantified 7 days post middle cerebral artery occlusion. Statistical analysis was conducted using the t-test. Results Qualitative RT-PCR revealed these proteases in ipsilateral and contralateral cortices. Dipeptidyl peptidase II and aminopeptidase N were up-regulated ipsilaterally from 6 h to 7 days post ischemia, whereas dipeptidyl peptidase 9 and cytosolic alanyl-aminopeptidase were transiently down-regulated at day 3. Dipeptidyl peptidase 8 and aminopeptidase N immunoreactivities were detected in cortical neurons of the contralateral hemisphere. At the same time point, dipeptidyl peptidase IV, 8 and aminopeptidase N were identified in activated microglia and macrophages in the ipsilateral cortex. Seven days post artery occlusion, dipeptidyl peptidase IV immunoreactivity was found in the perikarya of surviving cortical neurons of the ipsilateral hemisphere, whereas their nuclei were dipeptidyl peptidase 8- and amino peptidase N-positive. At the same time point, dipeptidyl peptidase IV, 8 and aminopeptidase N were targeted in astroglial cells. Total dipeptidyl peptidase IV, 8 and 9 activities remained constant in both hemispheres until day 3 post experimental ischemia, but were increased (+165%) in the ipsilateral cortex at day 7. In parallel, aminopeptidase N and cytosolic alanyl-aminopeptidase activities remained unchanged. Conclusions Distinct expression, localization and activity patterns of proline- and alanine-specific proteases indicate their involvement in ischemia-triggered inflammation and neurodegeneration. Consistently, IPC1755, a non-selective protease inhibitor, revealed a significant reduction of cortical lesions after transient cerebral ischemia and may suggest dipeptidyl peptidase IV, aminopeptidase N and proteases with similar substrate specificity as potentially therapy-relevant targets. PMID:22373413

Coronary veins determine the pattern of sympathetic innervation in the developing heart

PubMed Central

Nam, Joseph; Onitsuka, Izumi; Hatch, John; Uchida, Yutaka; Ray, Saugata; Huang, Siyi; Li, Wenling; Zang, Heesuk; Ruiz-Lozano, Pilar; Mukouyama, Yoh-suke

2013-01-01

Anatomical congruence of peripheral nerves and blood vessels is well recognized in a variety of tissues. Their physical proximity and similar branching patterns suggest that the development of these networks might be a coordinated process. Here we show that large diameter coronary veins serve as an intermediate template for distal sympathetic axon extension in the subepicardial layer of the dorsal ventricular wall of the developing mouse heart. Vascular smooth muscle cells (VSMCs) associate with large diameter veins during angiogenesis. In vivo and in vitro experiments demonstrate that these cells mediate extension of sympathetic axons via nerve growth factor (NGF). This association enables topological targeting of axons to final targets such as large diameter coronary arteries in the deeper myocardial layer. As axons extend along veins, arterial VSMCs begin to secrete NGF, which allows axons to reach target cells. We propose a sequential mechanism in which initial axon extension in the subepicardium is governed by transient NGF expression by VSMCs as they are recruited to coronary veins; subsequently, VSMCs in the myocardium begin to express NGF as they are recruited by remodeling arteries, attracting axons toward their final targets. The proposed mechanism underlies a distinct, stereotypical pattern of autonomic innervation that is adapted to the complex tissue structure and physiology of the heart. PMID:23462468

Changes in the expression of DNA-binding/differentiation protein inhibitors in neurons and glial cells of the gerbil hippocampus following transient global cerebral ischemia

PubMed Central

LEE, JAE-CHUL; CHEN, BAI HUI; CHO, JEONG-HWI; KIM, IN HYE; AHN, JI HYEON; PARK, JOON HA; TAE, HYUN-JIN; CHO, GEUM-SIL; YAN, BING CHUN; KIM, DAE WON; HWANG, IN KOO; PARK, JINSEU; LEE, YUN LYUL; CHOI, SOO YOUNG; WON, MOO-HO

2015-01-01

Inhibitors of DNA-binding/differentiation (ID) proteins bind to basic helix-loop-helix (bHLH) transcription factors, including those that regulate differentiation and cell-cycle progression during development, and regulate gene transcription. However, little is known about the role of ID proteins in the brain under transient cerebral ischemic conditions. In the present study, we examined the effects of ischemia-reperfusion (I-R) injury on the immunoreactivity and protein levels of IDs 1–4 in the gerbil hippocampus proper Cornu Ammonis regions CA1–3 following 5 min of transient cerebral ischemia. Strong ID1 immunoreactivity was detected in the nuclei of pyramidal neurons in the hippocampal CA1–3 regions; immunoreactivity was significantly changed following I-R in the CA1 region, but not in the CA2/3 region. Five days following I-R, ID1 immunoreactivity was not detected in the CA1 pyramidal neurons. ID1 immunoreactivity was detected only in GABAergic interneurons in the ischemic CA1 region. Weak ID4 immunoreactivity was detected in non-pyramidal cells, and immunoreactivity was again only changed in the ischemic CA1 region. Five days following I-R, strong ID4 immunoreactivity was detected in non-pyramidal cells, which were identified as microglia, and not astrocytes, in the ischemic CA1 region. Furthermore, changes in the protein levels of ID1 and ID4 in the ischemic CA1 region studied by western blot were consistent with patterns of immunoreactivity. In summary, these results indicate that immunoreactivity and protein levels of ID1 and ID4 are distinctively altered following transient cerebral ischemia only in the CA1 region, and that the changes in ID1 and ID4 expression may relate to the ischemia-induced delayed neuronal death. PMID:25503067

PEDF Is Associated with the Termination of Chondrocyte Phenotype and Catabolism of Cartilage Tissue.

PubMed

Klinger, P; Lukassen, S; Ferrazzi, F; Ekici, A B; Hotfiel, T; Swoboda, B; Aigner, T; Gelse, K

2017-01-01

Objective. To investigate the expression and target genes of pigment epithelium-derived factor (PEDF) in cartilage and chondrocytes, respectively. Methods. We analyzed the expression pattern of PEDF in different human cartilaginous tissues including articular cartilage, osteophytic cartilage, and fetal epiphyseal and growth plate cartilage, by immunohistochemistry and quantitative real-time (qRT) PCR. Transcriptome analysis after stimulation of human articular chondrocytes with rhPEDF was performed by RNA sequencing (RNA-Seq) and confirmed by qRT-PCR. Results. Immunohistochemically, PEDF could be detected in transient cartilaginous tissue that is prone to undergo endochondral ossification, including epiphyseal cartilage, growth plate cartilage, and osteophytic cartilage. In contrast, PEDF was hardly detected in healthy articular cartilage and in the superficial zone of epiphyses, regions that are characterized by a permanent stable chondrocyte phenotype. RNA-Seq analysis and qRT-PCR demonstrated that rhPEDF significantly induced the expression of a number of matrix-degrading factors including SAA1, MMP1, MMP3, and MMP13. Simultaneously, a number of cartilage-specific genes including COL2A1, COL9A2, COMP, and LECT were among the most significantly downregulated genes. Conclusions. PEDF represents a marker for transient cartilage during all neonatal and postnatal developmental stages and promotes the termination of cartilage tissue by upregulation of matrix-degrading factors and downregulation of cartilage-specific genes. These data provide the basis for novel strategies to stabilize the phenotype of articular cartilage and prevent its degradation.

Development of transgenic finger millet (Eleusine coracana (L.) Gaertn.) resistant to leaf blast disease.

PubMed

Ignacimuthu, S; Ceasar, S Antony

2012-03-01

Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R0 and R1 progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R1 lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance.

BIN1 is reduced and Cav1.2 trafficking is impaired in human failing cardiomyocytes.

PubMed

Hong, Ting-Ting; Smyth, James W; Chu, Kevin Y; Vogan, Jacob M; Fong, Tina S; Jensen, Brian C; Fang, Kun; Halushka, Marc K; Russell, Stuart D; Colecraft, Henry; Hoopes, Charles W; Ocorr, Karen; Chi, Neil C; Shaw, Robin M

2012-05-01

Heart failure is a growing epidemic, and a typical aspect of heart failure pathophysiology is altered calcium transients. Normal cardiac calcium transients are initiated by Cav1.2 channels at cardiac T tubules. Bridging integrator 1 (BIN1) is a membrane scaffolding protein that causes Cav1.2 to traffic to T tubules in healthy hearts. The mechanisms of Cav1.2 trafficking in heart failure are not known. To study BIN1 expression and its effect on Cav1.2 trafficking in failing hearts. Intact myocardium and freshly isolated cardiomyocytes from nonfailing and end-stage failing human hearts were used to study BIN1 expression and Cav1.2 localization. To confirm Cav1.2 surface expression dependence on BIN1, patch-clamp recordings were performed of Cav1.2 current in cell lines with and without trafficking-competent BIN1. Also, in adult mouse cardiomyocytes, surface Cav1.2 and calcium transients were studied after small hairpin RNA-mediated knockdown of BIN1. For a functional readout in intact heart, calcium transients and cardiac contractility were analyzed in a zebrafish model with morpholino-mediated knockdown of BIN1. BIN1 expression is significantly decreased in failing cardiomyocytes at both mRNA (30% down) and protein (36% down) levels. Peripheral Cav1.2 is reduced to 42% by imaging, and a biochemical T-tubule fraction of Cav1.2 is reduced to 68%. The total calcium current is reduced to 41% in a cell line expressing a nontrafficking BIN1 mutant. In mouse cardiomyocytes, BIN1 knockdown decreases surface Cav1.2 and impairs calcium transients. In zebrafish hearts, BIN1 knockdown causes a 75% reduction in calcium transients and severe ventricular contractile dysfunction. The data indicate that BIN1 is significantly reduced in human heart failure, and this reduction impairs Cav1.2 trafficking, calcium transients, and contractility. Copyright © 2012 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

BIN1 is Reduced and Cav1.2 Trafficking is Impaired in Human Failing Cardiomyocytes

PubMed Central

Hong, Ting-Ting; Smyth, James W.; Chu, Kevin Y.; Vogan, Jacob M.; Fong, Tina S.; Jensen, Brian C.; Fang, Kun; Halushka, Marc K.; Russell, Stuart D.; Colecraft, Henry; Hoopes, Charles W.; Ocorr, Karen; Chi, Neil C.; Shaw, Robin M.

2011-01-01

Background Heart failure is a growing epidemic and a typical aspect of heart failure pathophysiology is altered calcium transients. Normal cardiac calcium transients are initiated by Cav1.2 channels at cardiac T-tubules. BIN1 is a membrane scaffolding protein that causes Cav1.2 to traffic to T-tubules in healthy hearts. The mechanisms of Cav1.2 trafficking in heart failure are not known. Objective To study BIN1 expression and its effect on Cav1.2 trafficking in failing hearts. Methods Intact myocardium and freshly isolated cardiomyocytes from non-failing and end-stage failing human hearts were used to study BIN1 expression and Cav1.2 localization. To confirm Cav1.2 surface expression dependence on BIN1, patch clamp recordings were performed of Cav1.2 current in cell lines with and without trafficking competent BIN1. Also, in adult mouse cardiomyocytes, surface Cav1.2 and calcium transients were studied after shRNA mediated knockdown of BIN1. For a functional readout in intact heart, calcium transients and cardiac contractility were analyzed in a zebrafish model with morpholino mediated knockdown of BIN1. Results BIN1 expression is significantly decreased in failing cardiomyocytes at both mRNA (30% down) and protein (36% down) levels. Peripheral Cav1.2 is reduced 42% by imaging and biochemical T-tubule fraction of Cav1.2 is reduced 68%. Total calcium current is reduced 41% in a cell line expressing non-trafficking BIN1 mutant. In mouse cardiomyocytes, BIN1 knockdown decreases surface Cav1.2 and impairs calcium transients. In zebrafish hearts, BIN1 knockdown causes a 75% reduction in calcium transients and severe ventricular contractile dysfunction. Conclusions The data indicate that BIN1 is significantly reduced in human heart failure, and this reduction impairs Cav1.2 trafficking, calcium transients, and contractility. PMID:22138472

Transient storage assessments of dye-tracer injections in rivers of the Willamette Basin, Oregon

USGS Publications Warehouse

Laenen, A.; Bencala, K.E.

2001-01-01

Rhodamine WT dye-tracer injections in rivers of the Willamette Basin yield concentration-time curves with characteristically long recession times suggestive of active transient storage processes. The scale of drainage areas contributing to the stream reaches studied in the Willamette Basin ranges from 10 to 12,000 km2. A transient storage assessment of the tracer studies has been completed using the U.S. Geological Survey's One-dimensional Transport with Inflow and Storage (OTIS) model, which incorporates storage exchange and decay functions along with the traditional dispersion and advection transport equation. The analysis estimates solute transport of the dye. It identifies first-order decay coefficients to be on the order of 10-5/sec for the nonconservative Rhodamine WT. On an individual subreach basis, the first-order decay is slower (typically by an order of magnitude) than the transient storage process, indicating that nonconservative tracers may be used to evaluate transient storage in rivers. In the transient storage analysis, a dimensionless parameter (As/A) expresses the spatial extent of storage zone area relative to stream cross section. In certain reaches of Willamette Basin pool-and-riffle, gravel-bed rivers, this parameter was as large as 0.5. A measure of the storage exchange flux was calculated for each stream subreach in the simulation analysis. This storage exchange is shown subjectively to be higher at higher stream discharges. Hyporheic linkage between streams and subsurface flows is the probable physical mechanism contributing to a significant part of this inferred active transient storage. Hyporheic linkages are further suggested by detailed measurements of river discharge with an Acoustic Doppler Current Profiler system delineating zones in two large rivers where water alternately enters and leaves the surface channels through graveland-cobble riverbeds. Measurements show patterns of hyporheic exchange that are highly variable in time and space.

EEG patterns in theta and gamma frequency range and their probable relation to human voluntary movement organization.

PubMed

Popivanov, D; Mineva, A; Krekule, I

1999-05-21

In experiments with EEG accompanying continuous slow goal-directed voluntary movements we found abrupt short-term transients (STs) of the coefficients of EEG time-varying autoregressive (TVAR) model. The onset of STs indicated (i) a positive EEG wave related to an increase of 3-7 Hz oscillations in time period before the movement start, (ii) synchronization of 35-40 Hz prior to movement start and during the movement when the target is nearly reached. Both these phenomena are expressed predominantly over supplementary motor area, premotor and parietal cortices. These patterns were detected after averaging of EEG segments synchronized to the abrupt changes of the TVAR coefficients computed in the time course of EEG single records. The results are discussed regarding the cognitive aspect of organization of goal-directed movements.

Genomic Organization, Phylogenetic and Expression Analysis of the B-BOX Gene Family in Tomato

PubMed Central

Chu, Zhuannan; Wang, Xin; Li, Ying; Yu, Huiyang; Li, Jinhua; Lu, Yongen; Li, Hanxia; Ouyang, Bo

2016-01-01

The B-BOX (BBX) proteins encode a class of zinc-finger transcription factors possessing one or two B-BOX domains and in some cases an additional CCT (CO, CO-like and TOC1) motif, which play important roles in regulating plant growth, development and stress response. Nevertheless, no systematic study of BBX genes has undertaken in tomato (Solanum lycopersicum). Here we present the results of a genome-wide analysis of the 29 BBX genes in this important vegetable species. Their structures, conserved domains, phylogenetic relationships, subcellular localizations, and promoter cis-regulatory elements were analyzed; their tissue expression profiles and expression patterns under various hormones and stress treatments were also investigated in detail. Tomato BBX genes can be divided into five subfamilies, and twelve of them were found to be segmentally duplicated. Real-time quantitative PCR analysis showed that most BBX genes exhibited different temporal and spatial expression patterns. The expression of most BBX genes can be induced by drought, polyethylene glycol-6000 or heat stress. Some BBX genes were induced strongly by phytohormones such as abscisic acid, gibberellic acid, or ethephon. The majority of tomato BBX proteins was predicted to be located in nuclei, and the transient expression assay using Arabidopsis mesophyll protoplasts demonstrated that all the seven BBX members tested (SlBBX5, 7, 15, 17, 20, 22, and 24) were localized in nucleus. Our analysis of tomato BBX genes on the genome scale would provide valuable information for future functional characterization of specific genes in this family. PMID:27807440

Developmental and Cell-Specific Expression of Thyroid Hormone Transporters in the Mouse Cochlea

PubMed Central

Sharlin, David S.; Visser, Theo J.

2011-01-01

Thyroid hormone is essential for the development of the cochlea and auditory function. Cochlear response tissues, which express thyroid hormone receptor β (encoded by Thrb), include the greater epithelial ridge and sensory epithelium residing inside the bony labyrinth. However, these response tissues lack direct blood flow, implying that mechanisms exist to shuttle hormone from the circulation to target tissues. Therefore, we investigated expression of candidate thyroid hormone transporters L-type amino acid transporter 1 (Lat1), monocarboxylate transporter (Mct)8, Mct10, and organic anion transporting polypeptide 1c1 (Oatp1c1) in mouse cochlear development by in situ hybridization and immunofluorescence analysis. L-type amino acid transporter 1 localized to cochlear blood vessels and transiently to sensory hair cells. Mct8 localized to the greater epithelial ridge, tympanic border cells underlying the sensory epithelium, spiral ligament fibrocytes, and spiral ganglion neurons, partly overlapping with the Thrb expression pattern. Mct10 was detected in a highly restricted pattern in the outer sulcus epithelium and weakly in tympanic border cells and hair cells. Organic anion transporting polypeptide 1c1 localized primarily to fibrocytes in vascularized tissues of the spiral limbus and spiral ligament and to tympanic border cells. Investigation of hypothyroid Tshr−/− mice showed that transporter expression was delayed consistent with retardation of cochlear tissue maturation but not with compensatory responses to hypothyroidism. The results demonstrate specific expression of thyroid hormone transporters in the cochlea and suggest that a network of thyroid hormone transport underlies cochlear development. PMID:21878515

Air Exposure Induced Characteristics of Dry Eye in Conjunctival Tissue Culture

PubMed Central

Lin, Hui; Qu, Yangluowa; Geng, Zhixin; Li, Cheng; Wu, Huping; Dong, Nuo; Liu, Zuguo; Li, Wei

2014-01-01

There are several animal models illustrating dry eye pathophysiology. Current study would like to establish an ex vivo tissue culture model for characterizing dry eye. Human conjunctival explants were cultured under airlift or submerged conditions for up to 2 weeks, and only airlifted conjunctival cultures underwent increased epithelial stratification. Starting on day 4, the suprabasal cells displayed decreased K19 expression whereas K10 keratin became evident in airlift group. Pax6 nuclear expression attenuated already at 2 days, while its perinuclear and cytoplasmic expression gradually increased. MUC5AC and MUC19 expression dramatically decreased whereas the full thickness MUC4 and MUC16 expression pattern disappeared soon after initiating the airlift condition. Real time PCR showed K16, K10 and MUC16 gene up-regulated while K19, MUC5AC, MUC19 and MUC4 down-regulated on day 8 and day 14. On day 2 was the appearance of apoptotic epithelial and stromal cells appeared. The Wnt signaling pathway was transiently activated from day 2 to day 10. The inflammatory mediators IL-1β, TNF-α, and MMP-9 were detected in the conditioned media after 6 to 8 days. In conclusion, airlifted conjunctival tissue cultures demonstrated Wnt signaling pathway activation, coupled with squamous metaplasia, mucin pattern alteration, apoptosis and upregulation of proinflammatory cytokine expression. These changes mimic the pathohistological alterations described in dry eye. This correspondence suggests that insight into the pathophysiology of dry eye may be aided through the use of airlifted conjunctival tissue cultures. PMID:24498087

Delayed hippocampal neuronal death in young gerbil following transient global cerebral ischemia is related to higher and longer-term expression of p63 in the ischemic hippocampus

PubMed Central

Bae, Eun Joo; Chen, Bai Hui; Yan, Bing Chun; Shin, Bich Na; Cho, Jeong Hwi; Kim, In Hye; Ahn, Ji Hyeon; Lee, Jae Chul; Tae, Hyun-Jin; Hong, Seongkweon; Kim, Dong Won; Cho, Jun Hwi; Lee, Yun Lyul; Won, Moo-Ho; Park, Joon Ha

2015-01-01

The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions (CA1–3) between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia. p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group. p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults. PMID:26199612

The HMX/NKX homeodomain protein MLS-2 specifies the identity of the AWC sensory neuron type via regulation of the ceh-36 Otx gene in C. elegans

PubMed Central

Kim, Kyuhyung; Kim, Rinho; Sengupta, Piali

2010-01-01

The differentiated features of postmitotic neurons are dictated by the expression of specific transcription factors. The mechanisms by which the precise spatiotemporal expression patterns of these factors are regulated are poorly understood. In C. elegans, the ceh-36 Otx homeobox gene is expressed in the AWC sensory neurons throughout postembryonic development, and regulates terminal differentiation of this neuronal subtype. Here, we show that the HMX/NKX homeodomain protein MLS-2 regulates ceh-36 expression specifically in the AWC neurons. Consequently, the AWC neurons fail to express neuron type-specific characteristics in mls-2 mutants. mls-2 is expressed transiently in postmitotic AWC neurons, and directly initiates ceh-36 expression. CEH-36 subsequently interacts with a distinct site in its cis-regulatory sequences to maintain its own expression, and also directly regulates the expression of AWC-specific terminal differentiation genes. We also show that MLS-2 acts in additional neuron types to regulate their development and differentiation. Our analysis describes a transcription factor cascade that defines the unique postmitotic characteristics of a sensory neuron subtype, and provides insights into the spatiotemporal regulatory mechanisms that generate functional diversity in the sensory nervous system. PMID:20150279

Contribution of Egr1/zif268 to Activity-Dependent Arc/Arg3.1 Transcription in the Dentate Gyrus and Area CA1 of the Hippocampus

PubMed Central

Penke, Zsuzsa; Chagneau, Carine; Laroche, Serge

2011-01-01

Egr1, a member of the Egr family of transcription factors, and Arc are immediate early genes known to play major roles in synaptic plasticity and memory. Despite evidence that Egr family members can control Arc transcriptional regulation, demonstration of a selective role of Egr1 alone is lacking. We investigated the extent to which activity-dependent Arc expression is dependent on Egr1 by analyzing Arc mRNA expression using fluorescence in situ hybridization in the dorsal dentate gyrus and CA1 of wild-type (WT) and Egr1 knockout mice. Following electroconvulsive shock, we found biphasic expression of Arc in area CA1 in mice, consisting in a rapid (30 min) and transient wave followed by a second late-phase of expression (8 h), and a single but prolonged wave of expression in the dentate gyrus. Egr1 deficiency abolished the latest, but not the early wave of Arc expression in CA1, and curtailed that of the dentate gyrus. Since the early wave of Arc expression was not affected in Egr1 mutant mice, we next analyzed behaviorally induced Arc expression patterns as an index of neural ensemble activation in the dentate gyrus and area CA1 of WT and Egr1 mutant mice. Spatial exploration of novel or familiar environments induced in mice a single early and transient wave of Arc expression in the dentate gyrus and area CA1, which were not affected in Egr1 mutant mice. Analyses of Arc-expressing cells revealed that exploration recruits similar size dentate gyrus and CA1 neural ensembles in WT and Egr1 knockout mice. These findings suggest that hippocampal neural ensembles are normally activated immediately following spatial exploration in Egr1 knockout mice, indicating normal hippocampal encoding of information. They also provide evidence that in condition of strong activation Egr1 alone can control late-phases of activity-dependent Arc transcription in the dentate gyrus and area CA1 of the hippocampus. PMID:21887136

Intraluminal pressure patterns in the human colon assessed by high-resolution manometry

PubMed Central

Chen, Ji-Hong; Yu, Yuanjie; Yang, Zixian; Yu, Wen-Zhen; Chen, Wu Lan; Yu, Hui; Kim, Marie Jeong-Min; Huang, Min; Tan, Shiyun; Luo, Hesheng; Chen, Jianfeng; Chen, Jiande D. Z.; Huizinga, Jan D.

2017-01-01

Assessment of colonic motor dysfunction is rarely done because of inadequate methodology and lack of knowledge about normal motor patterns. Here we report on elucidation of intraluminal pressure patterns using High Resolution Colonic Manometry during a baseline period and in response to a meal, in 15 patients with constipation, chronically dependent on laxatives, 5 healthy volunteers and 9 patients with minor, transient, IBS-like symptoms but no sign of constipation. Simultaneous pressure waves (SPWs) were the most prominent propulsive motor pattern, associated with gas expulsion and anal sphincter relaxation, inferred to be associated with fast propagating contractions. Isolated pressure transients occurred in most sensors, ranging in amplitude from 5–230 mmHg. Rhythmic haustral boundary pressure transients occurred at sensors about 4–5 cm apart. Synchronized haustral pressure waves, covering 3–5 cm of the colon occurred to create a characteristic intrahaustral cyclic motor pattern at 3–6 cycles/min, propagating in mixed direction. This activity abruptly alternated with erratic patterns resembling the segmentation motor pattern of the small intestine. High amplitude propagating pressure waves (HAPWs) were too rare to contribute to function assessment in most subjects. Most patients, dependent on laxatives for defecation, were able to generate normal motor patterns in response to a meal. PMID:28216670

Cloning and Functional Characterization of a β-Pinene Synthase from Artemisia annua That Shows a Circadian Pattern of Expression1

PubMed Central

Lu, Shan; Xu, Ran; Jia, Jun-Wei; Pang, Jihai; Matsuda, Seiichi P.T.; Chen, Xiao-Ya

2002-01-01

Artemisia annua plants produce a broad range of volatile compounds, including monoterpenes, which contribute to the characteristic fragrance of this medicinal species. A cDNA clone, QH6, contained an open reading frame encoding a 582-amino acid protein that showed high sequence identity to plant monoterpene synthases. The prokaryotically expressed QH6 fusion protein converted geranyl diphosphate to (−)-β-pinene and (−)-α-pinene in a 94:6 ratio. QH6 was predominantly expressed in juvenile leaves 2 weeks postsprouting. QH6 transcript levels were transiently reduced following mechanical wounding or fungal elicitor treatment, suggesting that this gene is not directly involved in defense reaction induced by either of these treatments. Under a photoperiod of 12 h/12 h (light/dark), the abundance of QH6 transcripts fluctuated in a diurnal pattern that ebbed around 3 h before daybreak (9th h in the dark phase) and peaked after 9 h in light (9th h in the light phase). The contents of (−)-β-pinene in juvenile leaves and in emitted volatiles also varied in a diurnal rhythm, correlating strongly with mRNA accumulation. When A. annua was entrained by constant light or constant dark conditions, QH6 transcript accumulation continued to fluctuate with circadian rhythms. Under constant light, advanced cycles of fluctuation of QH6 transcript levels were observed, and under constant dark, the cycle was delayed. However, the original diurnal pattern could be regained when the plants were returned to the normal light/dark (12 h/12 h) photoperiod. This is the first report that monoterpene biosynthesis is transcriptionally regulated in a circadian pattern. PMID:12226526

Circadian gene expression regulates pulsatile gonadotropin-releasing hormone (GnRH) secretory patterns in the hypothalamic GnRH-secreting GT1-7 cell line.

PubMed

Chappell, Patrick E; White, Rachel S; Mellon, Pamela L

2003-12-03

Although it has long been established that episodic secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus is required for normal gonadotropin release, the molecular and cellular mechanisms underlying the synchronous release of GnRH are primarily unknown. We used the GT1-7 mouse hypothalamic cell line as a model for GnRH secretion, because these cells release GnRH in a pulsatile pattern similar to that observed in vivo. To explore possible molecular mechanisms governing secretory timing, we investigated the role of the molecular circadian clock in regulation of GnRH secretion. GT1-7 cells express many known core circadian clock genes, and we demonstrate that oscillations of these components can be induced by stimuli such as serum and the adenylyl cyclase activator forskolin, similar to effects observed in fibroblasts. Strikingly, perturbation of circadian clock function in GT1-7 cells by transient expression of the dominant-negative Clock-Delta19 gene disrupts normal ultradian patterns of GnRH secretion, significantly decreasing mean pulse frequency. Additionally, overexpression of the negative limb clock gene mCry1 in GT1-7 cells substantially increases GnRH pulse amplitude without a commensurate change in pulse frequency, demonstrating that an endogenous biological clock is coupled to the mechanism of neurosecretion in these cells and can regulate multiple secretory parameters. Finally, mice harboring a somatic mutation in the Clock gene are subfertile and exhibit a substantial increase in estrous cycle duration as revealed by examination of vaginal cytology. This effect persists in normal light/dark (LD) cycles, suggesting that a suprachiasmatic nucleus-independent endogenous clock in GnRH neurons is required for eliciting normal pulsatile patterns of GnRH secretion.

Resveratrol protects primary cortical neuron cultures from transient oxygen-glucose deprivation by inhibiting MMP-9.

PubMed

Gao, Dakuan; Huang, Tao; Jiang, Xiaofan; Hu, Shijie; Zhang, Lei; Fei, Zhou

2014-06-01

It was recently shown that resveratrol exerts neuroprotective effects against cerebral ischemia in mice. The aim of the present study was to further confirm these effects in in vitro primary cortical neuron cultures with transient oxygen-glucose deprivation (OGD), and to investigate whether these effects are due to the inhibition of matrix metalloproteinase-9 (MMP-9) and of cell apoptosis. Neuronal primary cultures of cerebral cortex were prepared from BALB/c mice embryos (13-15 days). Cells from 14- to 16-day cultures were subjected to OGD for 3 h, followed by 21 h of reoxygenation to simulate transient ischemia. Different doses of resveratrol were added into the culture medium during the simulation of transient ischemia. The effect of the extracellular signal-regulated kinase (ERK) inhibitor U0126 was studied by adding U0126 (5 µg/µl, 4 µl) into the culture medium during transient ischemia; as a control, we used treatment of cells with 50 µM of resveratrol. Cell viability was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reduction assay. Cell apoptosis was assessed by flow cytometry. The effects of resveratrol on the expression of MMP-9 were analyzed by western blotting and reverse transcription-polymerase chain reaction (RT-PCR), while the levels of ERK, phosphorylated (p)-ERK, cleaved caspase-3, Bax and Bcl-2 were measured by western blotting. The results of the MTT assay showed that cell viability is significantly reduced by transient OGD. OGD induced cell apoptosis, the expression of Bax and the activation of caspase-3 and ERK, inhibited the expression of Bcl-2 and increased the expression of MMP-9, while these effects were reversed by treatment with resveratrol. The therapeutic efficacy of resveratrol was shown to be dose-dependent, with the most suitable dose range determined at 50-100 µM. Treatment with U0126 inhibited MMP-9 and Bax expression and caspase-3 activation, while it further promoted the expression of the anti-apoptotic molecule Bcl-2, suggesting that resveratrol inhibits MMP-9 expression and cell apoptosis by attenuating the activation of ERK1/2. In conclusion, OGD can induce apoptosis through canonical apoptotic signals and by regulating the expression of MMP-9; the anti-apoptotic activity of resveratrol and its inhibitory effect on MMP-9 expression contribute in the reduced activation of ERK.

Space Use and Habitat Selection by Resident and Transient Coyotes (Canis latrans)

PubMed Central

Hinton, Joseph W.; van Manen, Frank T.; Chamberlain, Michael J.

2015-01-01

Little information exists on coyote (Canis latrans) space use and habitat selection in the southeastern United States and most studies conducted in the Southeast have been carried out within small study areas (e.g., ≤1,000 km2). Therefore, studying the placement, size, and habitat composition of coyote home ranges over broad geographic areas could provide relevant insights regarding how coyote populations adjust to regionally varying ecological conditions. Despite an increasing number of studies of coyote ecology, few studies have assessed the role of transiency as a life-history strategy among coyotes. During 2009–2011, we used GPS radio-telemetry to study coyote space use and habitat selection on the Albemarle Peninsula of northeastern North Carolina. We quantified space use and 2nd- and 3rd-order habitat selection for resident and transient coyotes to describe space use patterns in a predominantly agricultural landscape. The upper limit of coyote home-range size was approximately 47 km2 and coyotes exhibiting shifting patterns of space use of areas >65 km2 were transients. Transients exhibited localized space use patterns for short durations prior to establishing home ranges, which we defined as “biding” areas. Resident and transient coyotes demonstrated similar habitat selection, notably selection of agricultural over forested habitats. However, transients exhibited stronger selection for roads than resident coyotes. Although transient coyotes are less likely to contribute reproductively to their population, transiency may be an important life history trait that facilitates metapopulation dynamics through dispersal and the eventual replacement of breeding residents lost to mortality. PMID:26148130

Space use and habitat selection by resident and transient coyotes (Canis latrans)

USGS Publications Warehouse

Hinton, Joseph W; van Manen, Frank T.; Chamberlain, Michael J

2015-01-01

Little information exists on coyote (Canis latrans) space use and habitat selection in the southeastern United States and most studies conducted in the Southeast have been carried out within small study areas (e.g., ≤1,000 km2). Therefore, studying the placement, size, and habitat composition of coyote home ranges over broad geographic areas could provide relevant insights regarding how coyote populations adjust to regionally varying ecological conditions. Despite an increasing number of studies of coyote ecology, few studies have assessed the role of transiency as a life-history strategy among coyotes. During 2009–2011, we used GPS radio-telemetry to study coyote space use and habitat selection on the Albemarle Peninsula of northeastern North Carolina. We quantified space use and 2nd- and 3rd-order habitat selection for resident and transient coyotes to describe space use patterns in a predominantly agricultural landscape. The upper limit of coyote home-range size was approximately 47 km2 and coyotes exhibiting shifting patterns of space use of areas >65 km2 were transients. Transients exhibited localized space use patterns for short durations prior to establishing home ranges, which we defined as “biding” areas. Resident and transient coyotes demonstrated similar habitat selection, notably selection of agricultural over forested habitats. However, transients exhibited stronger selection for roads than resident coyotes. Although transient coyotes are less likely to contribute reproductively to their population, transiency may be an important life history trait that facilitates metapopulation dynamics through dispersal and the eventual replacement of breeding residents lost to mortality.

An intronless form of the tobacco extensin gene terminator strongly enhances transient gene expression in plant leaves.

PubMed

Rosenthal, Sun Hee; Diamos, Andrew G; Mason, Hugh S

2018-03-01

We have found interesting features of a plant gene (extensin) 3' flanking region, including extremely efficient polyadenylation which greatly improves transient expression of transgenes when an intron is removed. Its use will greatly benefit studies of gene expression in plants, research in molecular biology, and applications for recombinant proteins. Plants are a promising platform for the production of recombinant proteins. To express high-value proteins in plants efficiently, the optimization of expression cassettes using appropriate regulatory sequences is critical. Here, we characterize the activity of the tobacco extensin (Ext) gene terminator by transient expression in Nicotiana benthamiana, tobacco, and lettuce. Ext is a member of the hydroxyproline-rich glycoprotein (HRGP) superfamily and constitutes the major protein component of cell walls. The present study demonstrates that the Ext terminator with its native intron removed increased transient gene expression up to 13.5-fold compared to previously established terminators. The enhanced transgene expression was correlated with increased mRNA accumulation and reduced levels of read-through transcripts, which could impair gene expression. Analysis of transcript 3'-ends found that the majority of polyadenylated transcripts were cleaved at a YA dinucleotide downstream from a canonical AAUAAA motif and a UG-rich region, both of which were found to be highly conserved among related extensin terminators. Deletion of either of these regions eliminated most of the activity of the terminator. Additionally, a 45 nt polypurine sequence ~ 175 nt upstream from the polyadenylation sites was found to also be necessary for the enhanced expression. We conclude that the use of Ext terminator has great potential to benefit the production of recombinant proteins in plants.

Early and late components of EEG delay activity correlate differently with scene working memory performance

PubMed Central

Ng, Kenneth; Reichert, Chelsea P.

2017-01-01

Sustained and elevated activity during the working memory delay period has long been considered the primary neural correlate for maintaining information over short time intervals. This idea has recently been reinterpreted in light of findings generated from multiple neural recording modalities and levels of analysis. To further investigate the sustained or transient nature of activity, the temporal-spectral evolution (TSE) of delay period activity was examined in humans with high density EEG during performance of a Sternberg working memory paradigm with a relatively long six second delay and with novel scenes as stimuli. Multiple analyses were conducted using different trial window durations and different baseline periods for TSE computation. Sensor level analyses revealed transient rather than sustained activity during delay periods. Specifically, the consistent finding among the analyses was that high amplitude activity encompassing the theta range was found early in the first three seconds of the delay period. These increases in activity early in the delay period correlated positively with subsequent ability to distinguish new from old probe scenes. Source level signal estimation implicated a right parietal region of transient early delay activity that correlated positively with working memory ability. This pattern of results adds to recent evidence that transient rather than sustained delay period activity supports visual working memory performance. The findings are discussed in relation to synchronous and desynchronous intra- and inter-regional neural transmission, and choosing an optimal baseline for expressing temporal-spectral delay activity change. PMID:29016657

Transient synchrony among populations of five foliage-feeding Lepidoptera

Treesearch

Maartje J. Klapwijk; Jonathan A. Walter; Anikó Hirka; György Csóka; Christer Björkman; Andrew M. Liebhold

2018-01-01

Studies of transient population dynamics have largely focused on temporal changes in dynamical behaviour, such as the transition between periods of stability and instability. This study explores a related dynamic pattern, namely transient synchrony during a 49-year period among populations of five sympatric species of forest insects that share host tree resources. The...

Identification of Secretory Odontoblasts Using DMP1-GFP Transgenic Mice

PubMed Central

Balic, Anamaria; Mina, Mina

2011-01-01

Terminal differentiation of odontoblasts from dental papilla is a long process involving several intermediate steps and changes in the transcriptional profile and expression of proteins secreted by cells in the odontoblast lineage. Transgenic mouse lines in which GFP expression is under the control of tissue-and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stages of differentiation along a lineage. Our previous studies showed utilization of pOBCol3.6GFP and pOBCol2.3GFP animals for identification of odontoblasts at early and late stages of polarization respectively. In the present study we used the DMP1-GFP transgenic animal as an experimental model to examine its expression during the differentiation of odontoblasts from progenitor cells in vivo and in vitro. Our observations showed that DMP1-GFP transgene is first activated in secretory/functional odontoblasts engaged in secretion of predentin and then transiently expressed at high levels in newly differentiated odontoblasts. Expression of DMP1-GFP was down-regulated in highly differentiated odontoblasts. The temporal and spatial pattern of expression of DMP1-GFP transgene closely mimics the expression of endogenous DMP1. This transgenic animal will facilitate studies of gene expression and biological functions in secretory/functional odontoblasts. PMID:21172466

Galectin-3 expression in hippocampal CA2 following transient forebrain ischemia and its inhibition by hypothermia or antiapoptotic agents

PubMed Central

Hisamatsu, Kenji; Kobayashi, Kazuhiro; Miyazaki, Tatsuhiko; Hirata, Akihiro; Hatano, Yuichiro; Tomita, Hiroyuki; Hara, Akira

2016-01-01

Recent evidence has suggested that the hippocampal CA2 region plays an important role in the recognition process. We have reported that ischemic damage in the hippocampal CA2 region following transient ischemia is caused by apoptosis, but the underlying mechanisms are still not clear. Galectin-3 is a β-galactosidase-binding lectin that is important in cell proliferation and apoptotic regulation. We have also reported that galectin-3 was expressed in activated microglia in the CA1 region 96 h after transient ischemia. The aim of this study is to determine the localization and time course of galectin-3 expression in the CA2 region following transient forebrain ischemia. Galectin-3 immunostaining was observed in both interior side of CA1 region and CA2 region in hippocampus 60 h after ischemic insult. At 66 h, galectin-3 was observed in the whole CA1 region adjacent to the CA2 region in the hippocampus. Both galectin-3 expression and neuronal cell death in the CA2 region were significantly inhibited by hypothermia and by apoptosis-inhibiting reagents. These results suggest that galectin-3 in the CA2 region is expressed independent of that in the CA1 region. Protection of the expression of galectin-3 in the CA2 region might contribute toward the survival of CA2 pyramidal neurons. PMID:26848998

Generation of kisspeptin-responsive GnRH neurons from human pluripotent stem cells.

PubMed

Poliandri, Ariel; Miller, Duncan; Howard, Sasha; Nobles, Muriel; Ruiz-Babot, Gerard; Harmer, Stephen; Tinker, Andrew; McKay, Tristan; Guasti, Leonardo; Dunkel, Leo

2017-05-15

GnRH neurons are fundamental for reproduction in all vertebrates, integrating all reproductive inputs. The inaccessibility of human GnRH-neurons has been a major impediment to studying the central control of reproduction and its disorders. Here, we report the efficient generation of kisspeptin responsive GnRH-secreting neurons by directed differentiation of human Embryonic Stem Cells and induced-Pluripotent Stem Cells derived from a Kallman Syndrome patient and a healthy family member. The protocol involves the generation of intermediate Neural Progenitor Cells (NPCs) through long-term Bone morphogenetic protein 4 inhibition, followed by terminal specification of these NPCs in media containing Fibroblast Growth Factor 8 and a NOTCH inhibitor. The resulting GnRH-expressing and -secreting neurons display a neuroendocrine gene expression pattern and present spontaneous calcium transients that can be stimulated by kisspeptin. These in vitro generated GnRH expressing cells provide a new resource for studying the molecular mechanisms underlying the development and function of GnRH neurons. Copyright © 2017 Elsevier B.V. All rights reserved.

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

PubMed Central

2009-01-01

Background In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. Results The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. Conclusion We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine. PMID:19930574

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus.

PubMed

Lombardi, Raffaele; Circelli, Patrizia; Villani, Maria Elena; Buriani, Giampaolo; Nardi, Luca; Coppola, Valentina; Bianco, Linda; Benvenuto, Eugenio; Donini, Marcello; Marusic, Carla

2009-11-20

In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine.

Xenobiotics and loss of cell adhesion drive distinct transcriptional outcomes by aryl hydrocarbon receptor signaling.

PubMed

Hao, Nan; Lee, Kian Leong; Furness, Sebastian G B; Bosdotter, Cecilia; Poellinger, Lorenz; Whitelaw, Murray L

2012-12-01

The aryl hydrocarbon receptor (AhR) is a signal-regulated transcription factor, which is canonically activated by the direct binding of xenobiotics. In addition, switching cells from adherent to suspension culture also activates the AhR, representing a nonxenobiotic, physiological activation of AhR signaling. Here, we show that the AhR is recruited to target gene enhancers in both ligand [isopropyl-2-(1,3-dithietane-2-ylidene)-2-[N-(4-methylthiazol-2-yl)carbamoyl]acetate (YH439)]-treated and suspension cells, suggesting a common mechanism of target gene induction between these two routes of AhR activation. However, gene expression profiles critically differ between xenobiotic- and suspension-activated AhR signaling. Por and Cldnd1 were regulated predominantly by ligand treatments, whereas, in contrast, ApoER2 and Ganc were regulated predominantly by the suspension condition. Classic xenobiotic-metabolizing AhR targets such as Cyp1a1, Cyp1b1, and Nqo1 were regulated by both ligand and suspension conditions. Temporal expression patterns of AhR target genes were also found to vary, with examples of transient activation, transient repression, or sustained alterations in expression. Furthermore, sequence analysis coupled with chromatin immunoprecipitation assays and reporter gene analysis identified a functional xenobiotic response element (XRE) in the intron 1 of the mouse Tiparp gene, which was also bound by hypoxia-inducible factor-1α during hypoxia and features a concatemer of four XRE cores (GCGTG). Our data suggest that this XRE concatemer site concurrently regulates the expression of both the Tiparp gene and its cis antisense noncoding RNA after ligand- or suspension-induced AhR activation. This work provides novel insights into how AhR signaling drives different transcriptional programs via the ligand versus suspension modes of activation.

Poststroke Inflammasome Expression and Regulation in the Peri-Infarct Area by Gonadal Steroids after Transient Focal Ischemia in the Rat Brain.

PubMed

Lammerding, Leoni; Slowik, Alexander; Johann, Sonja; Beyer, Cordian; Zendedel, Adib

2016-01-01

CNS ischemia results in locally confined and rapid tissue damage accompanied by a loss of neurons and their circuits. Early and time-delayed inflammatory responses are critical variables determining the extent of neural disintegration and regeneration. Inflammasomes are vital effectors in innate immunity. Their activation in brain-intrinsic immune cells contributes to ischemia-related brain damage. The steroids 17β-estradiol (E2) and progesterone (P) are neuroprotective and anti-inflammatory. Using a transient focal rat ischemic model, we evaluated the time response of different inflammasomes in the peri-infarct zone from the early to late phases after poststroke ischemia. We show that the different inflammasome complexes reveal a specific time-oriented sequential expression pattern with a maximum at approximately 24 h after the infarct. Within the limits of antibody availability, immunofluorescence labeling demonstrated that microglia and neurons are major sources of the locally activated inflammasomes NOD-like receptor protein-3 (NLRP3) and associated speck-like protein (ASC), respectively. E2 and P given for 24 h immediately after ischemia onset reduced hypoxia-induced mRNA expression of the inflammasomes NLRC4, AIM2 and ASC, and decreased the protein levels of ASC and NLRP3. In addition, mRNA protein levels of the cytokines interleukin-1β (IL1β), IL18 and TNFα were reduced by the steroids. The findings provide for the first time a detailed flow chart of hypoxia-driven inflammasome regulation in the peri-infarct cerebral cortex. Further, we demonstrate that E2 and P alleviate the expression of certain inflammasome components, sometimes in a hormone-specific way. Besides directly regulating other cellular neuroprotective pathways, the control of inflammasomes by these steroids might contribute to its neuroprotective potency. © 2015 S. Karger AG, Basel.

Systems Analysis of Early Host Gene Expression Provides Clues for Transient Mycobacterium avium ssp avium vs. Persistent Mycobacterium avium ssp paratuberculosis Intestinal Infections

PubMed Central

Khare, Sangeeta; Drake, Kenneth L.; Lawhon, Sara D.; Nunes, Jairo E. S.; Figueiredo, Josely F.; Rossetti, Carlos A.; Gull, Tamara; Everts, Robin E.; Lewin, Harris. A.; Adams, Leslie Garry

2016-01-01

It has long been a quest in ruminants to understand how two very similar mycobacterial species, Mycobacterium avium ssp. paratuberculosis (MAP) and Mycobacterium avium ssp. avium (MAA) lead to either a chronic persistent infection or a rapid-transient infection, respectively. Here, we hypothesized that when the host immune response is activated by MAP or MAA, the outcome of the infection depends on the early activation of signaling molecules and host temporal gene expression. To test our hypothesis, ligated jejuno-ileal loops including Peyer’s patches in neonatal calves were inoculated with PBS, MAP, or MAA. A temporal analysis of the host transcriptome profile was conducted at several times post-infection (0.5, 1, 2, 4, 8 and 12 hours). When comparing the transcriptional responses of calves infected with the MAA versus MAP, discordant patterns of mucosal expression were clearly evident, and the numbers of unique transcripts altered were moderately less for MAA-infected tissue than were mucosal tissues infected with the MAP. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis. Bayesian network modeling identified mechanistic genes, gene-to-gene relationships, pathways and Gene Ontologies (GO) biological processes that are involved in specific cell activation during infection. MAP and MAA had significant different pathway perturbation at 0.5 and 12 hours post inoculation. Inverse processes were observed between MAP and MAA response for epithelial cell proliferation, negative regulation of chemotaxis, cell-cell adhesion mediated by integrin and regulation of cytokine-mediated signaling. MAP inoculated tissue had significantly lower expression of phagocytosis receptors such as mannose receptor and complement receptors. This study reveals that perturbation of genes and cellular pathways during MAP infection resulted in host evasion by mucosal membrane barrier weakening to access entry in the ileum, inhibition of Ca signaling associated with decreased phagosome-lysosome fusion as well as phagocytosis inhibition, bias toward Th2 cell immune response accompanied by cell recruitment, cell proliferation and cell differentiation; leading to persistent infection. Contrarily, MAA infection was related to cellular responses associated with activation of molecular pathways that release chemicals and cytokines involved with containment of infection and a strong bias toward Th1 immune response, resulting in a transient infection. PMID:27653506

Systems Analysis of Early Host Gene Expression Provides Clues for Transient Mycobacterium avium ssp avium vs. Persistent Mycobacterium avium ssp paratuberculosis Intestinal Infections.

PubMed

Khare, Sangeeta; Drake, Kenneth L; Lawhon, Sara D; Nunes, Jairo E S; Figueiredo, Josely F; Rossetti, Carlos A; Gull, Tamara; Everts, Robin E; Lewin, Harris A; Adams, Leslie Garry

It has long been a quest in ruminants to understand how two very similar mycobacterial species, Mycobacterium avium ssp. paratuberculosis (MAP) and Mycobacterium avium ssp. avium (MAA) lead to either a chronic persistent infection or a rapid-transient infection, respectively. Here, we hypothesized that when the host immune response is activated by MAP or MAA, the outcome of the infection depends on the early activation of signaling molecules and host temporal gene expression. To test our hypothesis, ligated jejuno-ileal loops including Peyer's patches in neonatal calves were inoculated with PBS, MAP, or MAA. A temporal analysis of the host transcriptome profile was conducted at several times post-infection (0.5, 1, 2, 4, 8 and 12 hours). When comparing the transcriptional responses of calves infected with the MAA versus MAP, discordant patterns of mucosal expression were clearly evident, and the numbers of unique transcripts altered were moderately less for MAA-infected tissue than were mucosal tissues infected with the MAP. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis. Bayesian network modeling identified mechanistic genes, gene-to-gene relationships, pathways and Gene Ontologies (GO) biological processes that are involved in specific cell activation during infection. MAP and MAA had significant different pathway perturbation at 0.5 and 12 hours post inoculation. Inverse processes were observed between MAP and MAA response for epithelial cell proliferation, negative regulation of chemotaxis, cell-cell adhesion mediated by integrin and regulation of cytokine-mediated signaling. MAP inoculated tissue had significantly lower expression of phagocytosis receptors such as mannose receptor and complement receptors. This study reveals that perturbation of genes and cellular pathways during MAP infection resulted in host evasion by mucosal membrane barrier weakening to access entry in the ileum, inhibition of Ca signaling associated with decreased phagosome-lysosome fusion as well as phagocytosis inhibition, bias toward Th2 cell immune response accompanied by cell recruitment, cell proliferation and cell differentiation; leading to persistent infection. Contrarily, MAA infection was related to cellular responses associated with activation of molecular pathways that release chemicals and cytokines involved with containment of infection and a strong bias toward Th1 immune response, resulting in a transient infection.

Transient silencing of the KASII genes is feasible in Nicotiana benthamiana for metabolic engineering of wax ester composition

PubMed Central

Aslan, Selcuk; Hofvander, Per; Dutta, Paresh; Sitbon, Folke; Sun, Chuanxin

2015-01-01

The beta-ketoacyl-ACP synthase II (KASII) is an enzyme in fatty acid biosynthesis, catalyzing the elongation of 16:0-acyl carrier protein (ACP) to 18:0-ACP in plastids. Mutations in KASII genes in higher plants can lead to lethality, which makes it difficult to utilize the gene for lipid metabolic engineering. We demonstrated previously that transient expression of plastid-directed fatty acyl reductases and wax ester synthases could result in different compositions of wax esters. We hypothesized that changing the ratio between C16 (palmitoyl-compounds) and C18 (stearoyl-compounds) in the plastidic acyl-ACP pool by inhibition of KASII expression would change the yield and composition of wax esters via substrate preference of the introduced enzymes. Here, we report that transient inhibition of KASII expression by three different RNAi constructs in leaves of N. benthamiana results in almost complete inhibition of KASII expression. The transient RNAi approach led to a shift of carbon flux from a pool of C18 fatty acids to C16, which significantly increased wax ester production in AtFAR6-containing combinations. The results demonstrate that transient inhibition of KASII in vegetative tissues of higher plants enables metabolic studies towards industrial production of lipids such as wax esters with specific quality and composition. PMID:26063537

Brain selective transgene expression in zebrafish using an NRSE derived motif

PubMed Central

Bergeron, Sadie A.; Hannan, Markus C.; Codore, Hiba; Fero, Kandice; Li, Grace H.; Moak, Zachary; Yokogawa, Tohei; Burgess, Harold A.

2012-01-01

Transgenic technologies enable the manipulation and observation of circuits controlling behavior by permitting expression of genetically encoded reporter genes in neurons. Frequently though, neuronal expression is accompanied by transgene expression in non-neuronal tissues, which may preclude key experimental manipulations, including assessment of the contribution of neurons to behavior by ablation. To better restrict transgene expression to the nervous system in zebrafish larvae, we have used DNA sequences derived from the neuron-restrictive silencing element (NRSE). We find that one such sequence, REx2, when used in conjunction with several basal promoters, robustly suppresses transgene expression in non-neuronal tissues. Both in transient transgenic experiments and in stable enhancer trap lines, suppression is achieved without compromising expression within the nervous system. Furthermore, in REx2 enhancer trap lines non-neuronal expression can be de-repressed by knocking down expression of the NRSE binding protein RE1-silencing transcription factor (Rest). In one line, we show that the resulting pattern of reporter gene expression coincides with that of the adjacent endogenous gene, hapln3. We demonstrate that three common basal promoters are susceptible to the effects of the REx2 element, suggesting that this method may be useful for confining expression from many other promoters to the nervous system. This technique enables neural specific targeting of reporter genes and thus will facilitate the use of transgenic methods to manipulate circuit function in freely behaving larvae. PMID:23293587

Fiber type-specific afferent nerve activity induced by transient contractions of rat bladder smooth muscle in pathological states

PubMed Central

Kuga, Nahoko; Tanioka, Asao; Hagihara, Koichiro; Kawai, Tomoyuki

2017-01-01

Bladder smooth muscle shows spontaneous phasic contractions, which undergo a variety of abnormal changes depending on pathological conditions. How abnormal contractions affect the activity of bladder afferent nerves remains to be fully tested. In this study, we examined the relationship between transient increases in bladder pressure, representing transient contraction of bladder smooth muscle, and spiking patterns of bladder afferent fibers of the L6 dorsal root, in rat pathological models. All recordings were performed at a bladder pressure of approximately 10 cmH2O by maintaining the degree of bladder filling. In the cyclophosphamide-induced model, both Aδ and C fibers showed increased sensitivity to transient bladder pressure increases. In the prostaglandin E2-induced model, Aδ fibers, but not C fibers, specifically showed overexcitation that was time-locked with transient bladder pressure increases. These fiber type-specific changes in nerve spike patterns may underlie the symptoms of urinary bladder diseases. PMID:29267380

The Influences of Glycosylation on the Antigenicity, Immunogenicity, and Protective Efficacy of Ebola Virus GP DNA Vaccines

DTIC Science & Technology

2006-11-22

multiple muta- tions were not studied, (iii) a vaccinia virus (VACV)- T7 system was used for transient expression, (iv) pseudotyped retrovi- ruses were used...those studies produced little to no detectable GP1 or GP2 in the transient VACV- T7 expression assays, whereas in our studies with the DNA con- structs...type GP2 was detected in pseudotyped retroviruses, a result seemingly in conflict with these authors’ findings with the VACV- T7 expression. Although

Techno-economic analysis of horseradish peroxidase production using a transient expression system in Nicotiana benthamiana.

PubMed

Walwyn, David Richard; Huddy, Suzanne M; Rybicki, Edward P

2015-01-01

Despite the advantages of plant-based transient expression systems relative to microbial or mammalian cell systems, the commercial production of recombinant proteins using plants has not yet been achieved to any significant extent. One of the challenges has been the lack of published data on the costs of manufacture for products other than biopharmaceuticals. In this study, we report on the techno-economic analysis of the production of a standard commercial enzyme, namely, horseradish peroxidase (HRP), using a transient expression system in Nicotiana benthamiana. Based on the proven plant yield of 240 mg HRP/kg biomass, a biomass productivity of 15-kg biomass/m(2)/year and a process yield of 54 % (mg HRP product/mg HRP in biomass), it is apparent that HRP can be manufactured economically via transient expression in plants in a large-scale facility (>5 kg HRP/year). At this level, the process is competitive versus the existing technology (extraction of the enzyme from horseradish), and the product is of comparable or improved activity, containing only the preferred isoenzyme C. Production scale, protein yield and biomass productivity are found to be the most important determinants of overall viability.

The murine SP-C promoter directs type II cell-specific expression in transgenic mice.

PubMed

Glasser, Stephan W; Eszterhas, Susan K; Detmer, Emily A; Maxfield, Melissa D; Korfhagen, Thomas R

2005-04-01

Genomic DNA from the mouse pulmonary surfactant protein C (SP-C) gene was analyzed in transgenic mice to identify DNA essential for alveolar type II cell-specific expression. SP-C promoter constructs extending either 13 or 4.8 kb upstream of the transcription start site directed lung-specific expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In situ hybridization analysis demonstrated alveolar cell-specific expression in the lungs of adult transgenic mice, and the pattern of 4.8 SP-C-CAT expression during development paralleled that of the endogenous SP-C gene. With the use of deletion constructs, lung-specific, low-level CAT activity was detected in tissue assays of SP-C-CAT transgenic mice retaining 318 bp of the promoter. In transient and stable cell transfection experiments, the 4.8-kb SP-C promoter was 90-fold more active as a stably integrated gene. These findings indicate that 1) the 4.8-kb SP-C promoter is sufficient to direct cell-specific and developmental expression, 2) an enhancer essential for lung-specific expression maps to the proximal 318-bp promoter, and 3) the activity of the 4.8-kb SP-C promoter construct is highly dependent on its chromatin environment.

Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells.

PubMed

Buschmann, H; Green, P; Sambade, A; Doonan, J H; Lloyd, C W

2011-04-01

Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division. Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Double-labelling in interphase and mitosis enabled localization studies. We found that the transient transformation efficiency was highest when BY-2/Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle. Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

Expression patterns of histone deacetylases in experimental stroke and potential targets for neuroprotection.

PubMed

Chen, Yan-Ting; Zang, Xue-Feng; Pan, Jie; Zhu, Xiao-Lei; Chen, Fei; Chen, Zhi-Bin; Xu, Yun

2012-09-01

1. Histone deacetylase (HDAC) inhibitors exert neuroprotection in both cellular and animal models of ischaemic stroke. However, which HDAC isoform (or isoforms) mediates this beneficial effect has not yet been determined. 2. In the present study, gene levels of the HDAC isoforms were determined in the mouse cortex using reverse transcription-polymerase chain reaction (RT-PCR), whereas changes in the expression of individual zinc-dependent HDAC family members were evaluated by western blotting, 3, 12, 24 and 48 h after cerebral ischaemia induced by transient middle cerebral artery occlusion in male Kunming mice. 3. The HDAC isoforms HDAC1-11 were all expressed in the mouse cortex and differentially affected by cerebral ischaemia. Notably, there was a substantial increase in HDAC3, HDAC6 and HDAC11 expression during the early phases of experimental stroke, indicating their contribution to stroke pathogenesis. Furthermore, induction of HDAC3 and HDAC6 in cortical neurons by ischaemic stroke was confirmed in vivo and in vitro using double-labelled immunostaining and RT-PCR, respectively. Therefore, small hairpin (sh) RNAs were used to selectively knock down HDAC3 or HDAC6. This knockdown appreciably promoted the survival of cortical neurons subjected to oxygen and glucose deprivation. 4. The findings of the present study demonstrate the expression patterns of HDAC isoforms during experimental ischaemic stroke. Furthermore, HDAC3 and HDAC6 were identified as potential mediators in the neurotoxicity of ischaemic stroke, suggesting that specific therapeutic approaches may be considered according to HDAC subtype. © 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Wiley Publishing Asia Pty Ltd.

Statistical Emulation of Climate Model Projections Based on Precomputed GCM Runs*

DOE PAGES

Castruccio, Stefano; McInerney, David J.; Stein, Michael L.; ...

2014-02-24

The authors describe a new approach for emulating the output of a fully coupled climate model under arbitrary forcing scenarios that is based on a small set of precomputed runs from the model. Temperature and precipitation are expressed as simple functions of the past trajectory of atmospheric CO 2 concentrations, and a statistical model is fit using a limited set of training runs. The approach is demonstrated to be a useful and computationally efficient alternative to pattern scaling and captures the nonlinear evolution of spatial patterns of climate anomalies inherent in transient climates. The approach does as well as patternmore » scaling in all circumstances and substantially better in many; it is not computationally demanding; and, once the statistical model is fit, it produces emulated climate output effectively instantaneously. In conclusion, it may therefore find wide application in climate impacts assessments and other policy analyses requiring rapid climate projections.« less

Cerebroglycan: an integral membrane heparan sulfate proteoglycan that is unique to the developing nervous system and expressed specifically during neuronal differentiation

PubMed Central

1994-01-01

Heparan sulfate proteoglycans (HSPGs) are found on the surface of all adherent cells and participate in the binding of growth factors, extracellular matrix glycoproteins, cell adhesion molecules, and proteases and antiproteases. We report here the cloning and pattern of expression of cerebroglycan, a glycosylphosphatidylinositol (GPI)- anchored HSPG that is found in the developing rat brain (previously referred to as HSPG M13; Herndon, M. E., and A. D. Lander. 1990. Neuron. 4:949-961). The cerebroglycan core protein has a predicted molecular mass of 58.6 kD and five potential heparan sulfate attachment sites. Together with glypican (David, G., V. Lories, B. Decock, P. Marynen, J.-J. Cassiman, and H. Van den Berghe. 1990. J. Cell Biol. 111:3165-3176), it defines a family of integral membrane HSPGs characterized by GPI linkage and conserved structural motifs, including a pattern of 14 cysteine residues that is absolutely conserved. Unlike other known integral membrane HSPGs, including glypican and members of the syndecan family of transmembrane proteoglycans, cerebroglycan is expressed in only one tissue: the nervous system. In situ hybridization experiments at several developmental stages strongly suggest that cerebroglycan message is widely and transiently expressed by immature neurons, appearing around the time of final mitosis and disappearing after cell migration and axon outgrowth have been completed. These results suggest that cerebroglycan may fulfill a function related to the motile behaviors of developing neurons. PMID:8294498

The up-regulation of miR-300 in gastric cancer and its effects on cells malignancy

PubMed Central

Shen, Zhen; Li, Chunsheng; Zhang, Kai; Yu, Wei; Xiao, Huijie; Li, Bo; Liu, Tongjun

2015-01-01

Objective: In this study, we investigated the role of miR-300 in regulating cell proliferation and invasion of gastric cancer cells. Methods: MicroRNA and protein expression patterns were compared between gastric cancer tissue and normal tissue and between two different prognostic groups. The up-regulation of miR-300 was confirmed by real-time reverse transcription polymerase chain reaction and its expression was analyzed in AGS gastric cancer cells. Results: We observed that miR-300 expression was frequently and dramatically up-regulated in human gastric cancer tissues and cell lines compared with the matched adjacent normal tissues and cells. We further showed that transient and stable over-expression of miR-300 could promote cell proliferation and cell cycle progression. Moreover, p53, a key inhibitor of cell cycle, was verified as a direct target of miR-300, suggesting that miR-300 might promote gastric cancer cell proliferation and invasion by increasing p53 expression. Conclusion: Our findings indicated that miR-300 up-regulation might exert some sort of antagonistic function by targeting p53 in gastric cancer cell proliferation during gastric tumorigenesis. PMID:26221215

The up-regulation of miR-300 in gastric cancer and its effects on cells malignancy.

PubMed

Shen, Zhen; Li, Chunsheng; Zhang, Kai; Yu, Wei; Xiao, Huijie; Li, Bo; Liu, Tongjun

2015-01-01

In this study, we investigated the role of miR-300 in regulating cell proliferation and invasion of gastric cancer cells. MicroRNA and protein expression patterns were compared between gastric cancer tissue and normal tissue and between two different prognostic groups. The up-regulation of miR-300 was confirmed by real-time reverse transcription polymerase chain reaction and its expression was analyzed in AGS gastric cancer cells. We observed that miR-300 expression was frequently and dramatically up-regulated in human gastric cancer tissues and cell lines compared with the matched adjacent normal tissues and cells. We further showed that transient and stable over-expression of miR-300 could promote cell proliferation and cell cycle progression. Moreover, p53, a key inhibitor of cell cycle, was verified as a direct target of miR-300, suggesting that miR-300 might promote gastric cancer cell proliferation and invasion by increasing p53 expression. Our findings indicated that miR-300 up-regulation might exert some sort of antagonistic function by targeting p53 in gastric cancer cell proliferation during gastric tumorigenesis.

MiR-300 regulate the malignancy of breast cancer by targeting p53.

PubMed

Xu, Xiao-Heng; Li, Da-Wei; Feng, Hui; Chen, Hong-Mei; Song, Yan-Qiu

2015-01-01

In this study, we investigated the role of miR-300 in regulating cell proliferation and invasion of breast cancer (BC) cells. MicroRNA and protein expression patterns were compared between breast cancer tissue and normal tissue and between two different prognostic groups. The up-regulation of miR-300 was confirmed by real-time reverse transcription polymerase chain reaction and its expression was analyzed in MCF-7 breast cancer cells. We observed that miR-300 expression was frequently and dramatically up-regulated in human breast cancer tissues and cell lines compared with the matched adjacent normal tissues and cells. We further showed that transient and stable over-expression of miR-300 could promote cell proliferation and cell cycle progression. Moreover, p53, a key inhibitor of cell cycle, was verified as a direct target of miR-300, suggesting that miR-300 might promote breast cancer cell proliferation and invasion by regulating p53 expression. Our findings indicated that miR-300 up-regulation might exert some sort of antagonistic function by targeting p53 in breast cancer cell proliferation during breast tumorigenesis.

MiR-300 regulate the malignancy of breast cancer by targeting p53

PubMed Central

Xu, Xiao-Heng; Li, Da-Wei; Feng, Hui; Chen, Hong-Mei; Song, Yan-Qiu

2015-01-01

Objective: In this study, we investigated the role of miR-300 in regulating cell proliferation and invasion of breast cancer (BC) cells. Methods: MicroRNA and protein expression patterns were compared between breast cancer tissue and normal tissue and between two different prognostic groups. The up-regulation of miR-300 was confirmed by real-time reverse transcription polymerase chain reaction and its expression was analyzed in MCF-7 breast cancer cells. Results: We observed that miR-300 expression was frequently and dramatically up-regulated in human breast cancer tissues and cell lines compared with the matched adjacent normal tissues and cells. We further showed that transient and stable over-expression of miR-300 could promote cell proliferation and cell cycle progression. Moreover, p53, a key inhibitor of cell cycle, was verified as a direct target of miR-300, suggesting that miR-300 might promote breast cancer cell proliferation and invasion by regulating p53 expression. Conclusion: Our findings indicated that miR-300 up-regulation might exert some sort of antagonistic function by targeting p53 in breast cancer cell proliferation during breast tumorigenesis. PMID:26221232

Identification of differentially expressed lncRNAs involved in transient regeneration of the neonatal C57BL/6J mouse heart by next-generation high-throughput RNA sequencing.

PubMed

Chen, Yu-Mei; Li, Hua; Fan, Yi; Zhang, Qi-Jun; Li, Xing; Wu, Li-Jie; Chen, Zi-Jie; Zhu, Chun; Qian, Ling-Mei

2017-04-25

Previous studies have shown that mammalian cardiac tissue has a regenerative capacity. Remarkably, neonatal mice can regenerate their cardiac tissue for up to 6 days after birth, but this capacity is lost by day 7. In this study, we aimed to explore the expression pattern of long noncoding RNA (lncRNA) during this period and examine the mechanisms underlying this process. We found that 685 lncRNAs and 1833 mRNAs were differentially expressed at P1 and P7 by the next-generation high-throughput RNA sequencing. The coding genes associated with differentially expressed lncRNAs were mainly involved in metabolic processes and cell proliferation, and also were potentially associated with several key regeneration signalling pathways, including PI3K-Akt, MAPK, Hippo and Wnt. In addition, we identified some correlated targets of highly-dysregulated lncRNAs such as Igfbp3, Trnp1, Itgb6, and Pim3 by the coding-noncoding gene co-expression network. These data may offer a reference resource for further investigation about the mechanisms by which lncRNAs regulate cardiac regeneration.

Elevated Peritoneal Fluid TNF-α Incites Ovarian Early Growth Response Factor 1 Expression and Downstream Protease Mediators

PubMed Central

Birt, Julie A.; Nabli, Henda; Stilley, Julie A.; Windham, Emma A.; Frazier, Shellaine R.

2013-01-01

Endometriosis-associated infertility manifests itself via multiple, poorly understood mechanisms. Our goal was to characterize signaling pathways, between peritoneal endometriotic lesions and the ovary, leading to failed ovulation. Genome-wide microarray analysis comparing ovarian tissue from an in vivo endometriosis model in the rat (Endo) with controls (Sham) identified 22 differentially expressed genes, including transiently expressed early growth response factor 1 (Egr1). The Egr1 regulates gene requisites for ovulation. The Egr1 promoter is responsive to tumor necrosis factor-alpha (TNF-α) signaling. We hypothesized that altered expression of ovarian EGR1 is induced by elevated peritoneal fluid TNF-α which is upregulated by the presence of peritoneal endometriosis. Endo rats, compared to controls, had more peritoneal fluid TNF-α and quantitative, spatial differences in Egr1 mRNA and EGR1 protein localization in follicular compartments. Interactions between elevated peritoneal fluid TNF-α and overexpression of follicular Egr1/EGR1 expression may affect downstream protease pathways impeding ovulation in endometriosis. Preliminary studies identified similar patterns of EGR1 protein localization in human ovaries from women with endometriosis and compared to those without endometriosis. PMID:23427178

Small reduction of neurokinin-1 receptor-expressing neurons in the pre-Bötzinger complex area induces abnormal breathing periods in awake goats.

PubMed

Wenninger, J M; Pan, L G; Klum, L; Leekley, T; Bastastic, J; Hodges, M R; Feroah, T; Davis, S; Forster, H V

2004-11-01

In awake rats, >80% bilateral reduction of neurokinin-1 receptor (NK1R)-expressing neurons in the pre-Bötzinger complex (pre-BötzC) resulted in hypoventilation and an "ataxic" breathing pattern (Gray PA, Rekling JC, Bocchiaro CM, Feldman JL, Science 286: 1566-1568, 1999). Accordingly, the present study was designed to gain further insight into the role of the pre-BötzC area NK1R-expressing neurons in the control of breathing during physiological conditions. Microtubules were chronically implanted bilaterally into the medulla of adult goats. After recovery from surgery, the neurotoxin saporin conjugated to substance P, specific for NK1R-expressing neurons, was bilaterally injected (50 pM in 10 microl) into the pre-BötzC area during the awake state (n = 8). In unoperated goats, 34 +/- 0.01% of the pre-BötzC area neurons are immunoreactive for the NK1R, but, in goats after bilateral injection of SP-SAP into the pre-BötzC area, NK1R immunoreactivity was reduced to 22.5 +/- 2.5% (29% decrease, P < 0.01). Ten to fourteen days after the injection, the frequency of abnormal breathing periods was sixfold greater than before injection (107.8 +/- 21.8/h, P < 0.001). Fifty-six percent of these periods were breaths of varying duration and volume with an altered respiratory muscle activation pattern, whereas the remaining were rapid, complete breaths with coordinated inspiratory-expiratory cycles. The rate of occurrence and characteristics of abnormal breathing periods were not altered during a CO2 inhalation-induced hyperpnea. Pathological breathing patterns were eliminated during non-rapid eye movement sleep in seven of eight goats, but they frequently occurred on arousal from non-rapid eye movement sleep. We conclude that a moderate reduction in pre-BötzC NK1R-expressing neurons results in state-dependent transient changes in respiratory rhythm and/or eupneic respiratory muscle activation patterns.

Comparison of the expression of neurotransmitter and muscular genesis markers in the postnatal male mouse masseter and trigeminal ganglion during development.

PubMed

Kamata, Hiroaki; Karibe, Hiroyuki; Sato, Iwao

2018-06-01

Calcitonin gene-related peptide (CGRP) is released by motor neurons and affects skeletal muscle fiber and transient receptor potential cation channel subfamily V member 1 (TRPV1), an important marker of pain modulation. However, the expression of CGRP and TRPV1 in the trigeminal ganglion (TG) during changes and in feeding patterns has not been described. We used real-time reverse transcription polymerase chain reaction and in situ hybridization to investigate the mRNA expression levels of CGRP and TRPV1 in the TG. The expression of myosin heavy-chain (MyHC) isoforms was also investigated in the masseter muscle (MM) during the transition from sucking to mastication, an important functional trigger for muscle. The mRNA and protein levels of CGRP increased in the MM and TG from postnatal day 10 (P10) to P20 in male mice. The protein levels of TRPV1 were almost constant in the TG from P10 to P20, in contrast to increases in the MM. The mRNA abundance of TRPV1 in the TG and MM was increased from P10 to P20. The localization of an antisense probe was used to count CGRP cell numbers and found to differentiate the ophthalmic, maxillary, and mandibular nerve divisions of the TG. In particular, the number of CGRP + cells per 10,000 μm 2 in the maxillary and mandibular divisions of the TG gradually changed from P10 to P20. The expression of CGRP and TRPV1 in the TG and MM and the patterns of expression of different MyHC isoforms were affected by changes in feeding during male mouse development. © 2017 Wiley Periodicals, Inc.

Transcriptional regulation of arylalkylamine-N-acetyltransferase-2 gene in the pineal gland of the gilthead seabream.

PubMed

Zilberman-Peled, B; Appelbaum, L; Vallone, D; Foulkes, N S; Anava, S; Anzulovich, A; Coon, S L; Klein, D C; Falcón, J; Ron, B; Gothilf, Y

2007-01-01

Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebrafish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.

An LRR/Malectin Receptor-Like Kinase Mediates Resistance to Non-adapted and Adapted Powdery Mildew Fungi in Barley and Wheat

PubMed Central

Rajaraman, Jeyaraman; Douchkov, Dimitar; Hensel, Götz; Stefanato, Francesca L.; Gordon, Anna; Ereful, Nelzo; Caldararu, Octav F.; Petrescu, Andrei-Jose; Kumlehn, Jochen; Boyd, Lesley A.; Schweizer, Patrick

2016-01-01

Pattern recognition receptors (PRRs) belonging to the multigene family of receptor-like kinases (RLKs) are the sensing devices of plants for microbe- or pathogen-associated molecular patterns released from microbial organisms. Here we describe Rnr8 (for Required for non-host resistance 8) encoding HvLEMK1, a LRR-malectin domain-containing transmembrane RLK that mediates non-host resistance of barley to the non-adapted wheat powdery mildew fungus Blumeria graminis f.sp. tritici. Transgenic barley lines with silenced HvLEMK1 allow entry and colony growth of the non-adapted pathogen, although sporulation was reduced and final colony size did not reach that of the adapted barley powdery mildew fungus B. graminis f.sp. hordei. Transient expression of the barley or wheat LEMK1 genes enhanced resistance in wheat to the adapted wheat powdery mildew fungus while expression of the same genes did not protect barley from attack by the barley powdery mildew fungus. The results suggest that HvLEMK1 is a factor mediating non-host resistance in barley and quantitative host resistance in wheat to the wheat powdery mildew fungus. PMID:28018377

An LRR/Malectin Receptor-Like Kinase Mediates Resistance to Non-adapted and Adapted Powdery Mildew Fungi in Barley and Wheat.

PubMed

Rajaraman, Jeyaraman; Douchkov, Dimitar; Hensel, Götz; Stefanato, Francesca L; Gordon, Anna; Ereful, Nelzo; Caldararu, Octav F; Petrescu, Andrei-Jose; Kumlehn, Jochen; Boyd, Lesley A; Schweizer, Patrick

2016-01-01

Pattern recognition receptors (PRRs) belonging to the multigene family of receptor-like kinases (RLKs) are the sensing devices of plants for microbe- or pathogen-associated molecular patterns released from microbial organisms. Here we describe Rnr8 (for Required for non-host resistance 8 ) encoding HvLEMK1, a LRR-malectin domain-containing transmembrane RLK that mediates non-host resistance of barley to the non-adapted wheat powdery mildew fungus Blumeria graminis f.sp. tritici . Transgenic barley lines with silenced HvLEMK1 allow entry and colony growth of the non-adapted pathogen, although sporulation was reduced and final colony size did not reach that of the adapted barley powdery mildew fungus B. graminis f.sp. hordei . Transient expression of the barley or wheat LEMK1 genes enhanced resistance in wheat to the adapted wheat powdery mildew fungus while expression of the same genes did not protect barley from attack by the barley powdery mildew fungus. The results suggest that HvLEMK1 is a factor mediating non-host resistance in barley and quantitative host resistance in wheat to the wheat powdery mildew fungus.

Altered skeletal pattern of gene expression in response to spaceflight and hindlimb elevation

NASA Technical Reports Server (NTRS)

Bikle, D. D.; Harris, J.; Halloran, B. P.; Morey-Holton, E.

1994-01-01

Spaceflight leads to osteopenia, in part by inhibiting bone formation. Using an animal model (hindlimb elevation) that simulates the weightlessness of spaceflight, we and others showed a reversible inhibition of bone formation and bone mineralization. In this study, we have measured the mRNA levels of insulin-like growth factor I (IGF-I), IGF-I receptor (IGF-IR), alkaline phosphatase, and osteocalcin in the tibiae of rats flown aboard National Aeronautics and Space Administration Shuttle Flight STS-54 and compared the results with those obtained from their ground-based controls and from the bones of hindlimb-elevated animals. Spaceflight and hindlimb elevation transiently increase the mRNA levels for IGF-I, IGF-IR, and alkaline phosphatase but decrease the mRNA levels for osteocalcin. The changes in osteocalcin and alkaline phosphatase mRNA levels are consistent with a shift toward decreased maturation, whereas the rise in IGF-I and IGF-IR mRNA levels may indicate a compensatory response to the fall in bone formation. We conclude that skeletal unloading during spaceflight or hindlimb elevation resets the pattern of gene expression in the osteoblast, giving it a less mature profile.

Efficient production of antibody Fab fragment by transient gene expression in insect cells.

PubMed

Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

2017-08-01

Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

[Construction, identification and expression of three kinds of shuttle plasmids of adenovirus expression vector of hepatitis C virus structure gene].

PubMed

Cao, Yi-zhan; Hao, Chun-qiu; Feng, Zhi-hua; Zhou, Yong-xing; Li, Jin-ge; Jia, Zhan-sheng; Wang, Ping-zhong

2003-02-01

To construct three recombinant shuttle plasmids of adenovirus expression vector which can express hepatitis C virus(HCV) different structure genes(C, C+E1, C+E1+E2) in order to pack adenovirus expression vectors which can express HCV different structure gene effectively. The different HCV structure genes derived from the plasmid pBRTM/HCV1-3011 by using polymerase chain reaction (PCR) were inserted into the backward position of cytomegalovirus(CMV) immediate early promotor element of shuttle plasmid(pAd.CMV-Link.1) of adenovirus expression vector respectively, then the three recombinant plasmids (pAd.HCV-C, pAd.HCV-CE1, pAd.HCV-S) were obtained. The recombinant plasmids were identified by endonuclease, PCR and sequencing. HCV structure genes were expressed transiently with Lipofectamine 2000 coated in HepG2 cells which were confirmed by immunofluorescence and Western-Blot. Insert DNAs of the three recombinant plasmids' were confirmed to be HCV different structure genes by endonuclease, PCR and sequencing. The three recombinant plasmids can express HCV structure gene (C, C+E1, C+E1+E2) transiently in HepG2 cells which were confirmed by immunofluorescence and Western-Blot. The three recombinant shuttle plasmids of adenovirus expression vector can express HCV structure gene(C, C+E1, C+E1+E2) transiently. This should be useful to pack adenovirus expression vector which can express HCV structure genes.

Systemic Analysis of Heat Shock Response Induced by Heat Shock and a Proteasome Inhibitor MG132

PubMed Central

Kim, Hee-Jung; Joo, Hye Joon; Kim, Yung Hee; Ahn, Soyeon; Chang, Jun; Hwang, Kyu-Baek; Lee, Dong-Hee; Lee, Kong-Joo

2011-01-01

The molecular basis of heat shock response (HSR), a cellular defense mechanism against various stresses, is not well understood. In this, the first comprehensive analysis of gene expression changes in response to heat shock and MG132 (a proteasome inhibitor), both of which are known to induce heat shock proteins (Hsps), we compared the responses of normal mouse fibrosarcoma cell line, RIF- 1, and its thermotolerant variant cell line, TR-RIF-1 (TR), to the two stresses. The cellular responses we examined included Hsp expressions, cell viability, total protein synthesis patterns, and accumulation of poly-ubiquitinated proteins. We also compared the mRNA expression profiles and kinetics, in the two cell lines exposed to the two stresses, using microarray analysis. In contrast to RIF-1 cells, TR cells resist heat shock caused changes in cell viability and whole-cell protein synthesis. The patterns of total cellular protein synthesis and accumulation of poly-ubiquitinated proteins in the two cell lines were distinct, depending on the stress and the cell line. Microarray analysis revealed that the gene expression pattern of TR cells was faster and more transient than that of RIF-1 cells, in response to heat shock, while both RIF-1 and TR cells showed similar kinetics of mRNA expression in response to MG132. We also found that 2,208 genes were up-regulated more than 2 fold and could sort them into three groups: 1) genes regulated by both heat shock and MG132, (e.g. chaperones); 2) those regulated only by heat shock (e.g. DNA binding proteins including histones); and 3) those regulated only by MG132 (e.g. innate immunity and defense related molecules). This study shows that heat shock and MG132 share some aspects of HSR signaling pathway, at the same time, inducing distinct stress response signaling pathways, triggered by distinct abnormal proteins. PMID:21738571

Multi-tasking Sulf1/Sulf2 enzymes do not only facilitate extracellular cell signalling but also participate in cell cycle related nuclear events.

PubMed

Krishnakumar, Kavithanjali; Chakravorty, Ishani; Foy, Wendy; Allen, Steve; Justo, Tiago; Mukherjee, Abir; Dhoot, Gurtej K

2018-03-01

This study demonstrates highly dynamic spatial and temporal pattern of SULF1/SULF2 expression in a number of neuronal cell types growing in normal culture medium that included their transient nuclear mobilisation. Their nuclear translocation became particularly apparent during cell proliferation as both SULF1/SULF2 demonstrated not only cell membrane associated expression, their known site of function but also transient nuclear mobilisation during nuclear cell division. Nuclear localisation was apparent not only by immunocytochemical staining but also confirmed by immunoblotting staining of isolated nuclear fractions of C6, U87 and N2A cells. Immunocytochemical analysis demonstrated rapid nuclear exit of both SULF1/SULF2 following cell division that was slightly delayed but not blocked in a fraction of the polyploid cells observed in C6 cells. The overexpression of both Sulf1 and Sulf2 genes in C6 and U87 cells markedly promoted in vitro growth of these cells accompanied by nuclear mobilisation while inhibition of both these genes inhibited cell proliferation with little or no nuclear SULF1/SULF2 mobilisation. SULF1/SULF2 activity in these cells thus demonstrated a clear co-ordination of extracellular cell signalling with nuclear events related to cell proliferation. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

Intraspinal serotonergic neurons consist of two, temporally distinct populations in developing zebrafish

PubMed Central

Montgomery, Jacob E.; Wiggin, Timothy D.; Rivera-Perez, Luis M.; Lillesaar, Christina; Masino, Mark A.

2015-01-01

Zebrafish intraspinal serotonergic neuron (ISN) morphology and distribution have been examined in detail at different ages; however, some aspects of the development of these cells remain unclear. Although antibodies to serotonin (5-HT) have detected ISNs in the ventral spinal cord of embryos, larvae, and adults, the only tryptophan hydroxylase (tph) transcript that has been described in the spinal cord is tph1a. Paradoxically, spinal tph1a is expressed transiently in embryos, which brings the source of 5-HT in the ISNs of larvae and adults into question. Because the pet1 and tph2 promoters drive transgene expression in the spinal cord, we hypothesized that tph2 is expressed in spinal cords of zebrafish larvae. We confirmed this hypothesis through in situ hybridization. Next, we used 5-HT antibody labeling and transgenic markers of tph2-expressing neurons to identify a transient population of ISNs in embryos that was distinct from ISNs that appeared later in development. The existence of separate ISN populations may not have been recognized previously due to their shared location in the ventral spinal cord. Finally, we used transgenic markers and immunohistochemical labeling to identify the transient ISN population as GABAergic Kolmer-Agduhr double-prime (KA″) neurons. Altogether, this study revealed a novel developmental paradigm in which KA″ neurons are transiently serotonergic before the appearance of a stable population of tph2-expressing ISNs. PMID:26437856

Adenovirus-mediated gene transfer of pathogen-associated molecular patterns for cancer immunotherapy.

PubMed

Tosch, C; Geist, M; Ledoux, C; Ziller-Remi, C; Paul, S; Erbs, P; Corvaia, N; Von Hoegen, P; Balloul, J-M; Haegel, H

2009-04-01

The delivery of stimulatory signals to dendritic cells (DCs) in the tumor microenvironment could be an effective means to break tumor-induced tolerance. The work presented here evaluates the immunostimulatory properties of pathogen-associated molecular patterns (PAMPs), microbial molecules which bind Toll-like receptors and deliver activating signals to immune cells, when expressed in tumor cells using adenoviral (Ad) vectors. In vitro, transduction of A549 tumor cells with Ad vectors expressing either flagellin from Listeria monocytogenes or P40 protein from Klebsiella pneumoniae induced the maturation of human monocyte-derived DCs in co-cultures. In mixed lymphocyte reactions (MLRs), Ad-flagellin and Ad-P40 transduction of tumor cells stimulated lymphocyte proliferation and the secretion of IFN-gamma. In vivo, these vectors were used either as stand-alone immunoadjuvants injected intratumorally or as vaccine adjuvants combined with a tumor antigen-expressing vector. When Ad-PAMPs were administered intratumorally to mice bearing subcutaneous syngeneic B16F0-CAR (cocksackie-adenovirus receptor) melanomas, tumor progression was transiently inhibited by Ad-P40. In a therapeutic vaccine setting, the combination of Ad-MUC1 and Ad-PAMP vectors injected subcutaneously delayed the growth of implanted RenCa-MUC1 tumors and improved tumor rejection when compared with vaccination with Ad-MUC1 alone. These results suggest that Ad-PAMPs could be effective immunoadjuvants for cancer immunotherapy.

Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane

PubMed Central

2014-01-01

Background Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene. Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. Results The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences. Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures. Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes. In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. Conclusions We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane. PMID:24708613

Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane.

PubMed

Chou, Ting-Chun; Moyle, Richard L

2014-04-08

Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene.Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences.Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures.Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes.In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane.

Tissue plasminogen activator (tPA) as a reporter gene in transient gene expression.

PubMed

Cheng, S M; Lee, S G; Kalyan, N K; McCloud, S; Levner, M; Hung, P P

1987-01-01

Using the gene coding for tissue plasminogen activator (tPA) as a reporter gene, a transient gene expression system has been established. Vectors containing the full-length cDNA of tPA with its signal sequences were introduced into mammalian recipient cells by a modified gene transfer procedure. Thirty hours after transfection, the secreted tPA was found in serum-free medium and measured by a fibrin-agarose plate assay (FAPA). In this assay, tPA converts plasminogen into plasmin which then degrades high-Mr fibrin to produce cleared zones. The sizes of these zones correspond to quantities of tPA. The combination of transient tPA expression system and the FAPA provides a quick, sensitive, quantitative and non-destructive method to examine the strength of eukaryotic regulatory elements in tissue-culture cells.

Angiogenin Expression during Early Human Placental Development; Association with Blood Vessel Formation

PubMed Central

Pavlov, Nadine; Guibourdenche, Jean; Degrelle, Séverine A.; Evain-Brion, Danièle

2014-01-01

The placenta is a transient organ essential for fetal development. During human placental development, chorionic villi grow in coordination with a large capillary network resulting from both vasculogenesis and angiogenesis. Angiogenin is one of the most potent inducers of neovascularisation in experimental models in vivo. We and others have previously mapped angiogenin expression in the human term placenta. Here, we explored angiogenin involvement in early human placental development. We studied, angiogenin expression by in situ hybridisation and/or by RT-PCR in tissues and primary cultured trophoblastic cells and angiogenin cellular distribution by coimmunolabelling with cell markers: CD31 (PECAM-1), vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor-2 (VEGF-R2), Tie-2, von Willebrand factor, CD34, erythropoeitin receptor (Epo-R), alpha-smooth muscle actin, CD45, cytokeratin 7, and Ki-67. Extravillous and villous cytotrophoblasts, isolated and differentiated in vitro, expressed and secreted angiogenin. Angiogenin was detected in villous trophoblastic layers, and structured and nascent fetal vessels. In decidua, it was expressed by glandular epithelial cells, vascular cells and macrophages. The observed pattern of angiogenin expression is compatible with a role in blood vessel formation and in cross-talk between trophoblasts and endothelial cells. In view of angiogenin properties, we suggest that angiogenin may participate in placental vasculogenesis and organogenesis. PMID:25093183

Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos

PubMed Central

2012-01-01

Background Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish) and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38) in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff), contains three copies of oestrogen response elements (3ERE) that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS) elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein). Results The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1–2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2), the synthetic oestrogen 17α- ethinyloestradiol (EE2), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures). For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish). We also demonstrate that our construct works in medaka, another model fish test species, suggesting the transient assay is applicable for testing oestrogenic chemicals in fish generally. Conclusion Our results indicate that the transient expression assay system can be used as a rapid integrated testing system for environmental oestrogens and to detect the oestrogenic target sites in developing fish embryos. PMID:22726887

Transient SNAIL1 expression is necessary for metastatic competence in breast cancer.

PubMed

Tran, Hung D; Luitel, Krishna; Kim, Michael; Zhang, Kun; Longmore, Gregory D; Tran, David D

2014-11-01

SNAIL1 has been suggested to regulate breast cancer metastasis based on analyses of human breast tumor transcriptomes and experiments using cancer cell lines and xenografts. However, in vivo genetic experimental support for a role for SNAIL1 in breast cancer metastasis that develops in an immunocompetent tumor microenvironment has not been determined. To address this question, we created a genetic SNAIL1 model by coupling an endogenous SNAIL1 reporter with an inducible SNAIL1 transgene. Using multiple genetic models of breast cancer, we demonstrated that endogenous SNAIL1 expression was restricted to primary tumors that ultimately disseminate. SNAIL1 gene deletion either during the premalignant phase or after primary tumors have reached a palpable size blunted metastasis, indicating that late metastasis was the main driver of metastasis and that this was dependent on SNAIL1. Importantly, SNAIL1 expression during breast cancer metastasis was transient and forced transient, but not continuous. SNAIL1 expression in breast tumors was sufficient to increase metastasis. ©2014 American Association for Cancer Research.

Quantitative characterization of genetic parts and circuits for plant synthetic biology.

PubMed

Schaumberg, Katherine A; Antunes, Mauricio S; Kassaw, Tessema K; Xu, Wenlong; Zalewski, Christopher S; Medford, June I; Prasad, Ashok

2016-01-01

Plant synthetic biology promises immense technological benefits, including the potential development of a sustainable bio-based economy through the predictive design of synthetic gene circuits. Such circuits are built from quantitatively characterized genetic parts; however, this characterization is a significant obstacle in work with plants because of the time required for stable transformation. We describe a method for rapid quantitative characterization of genetic plant parts using transient expression in protoplasts and dual luciferase outputs. We observed experimental variability in transient-expression assays and developed a mathematical model to describe, as well as statistical normalization methods to account for, this variability, which allowed us to extract quantitative parameters. We characterized >120 synthetic parts in Arabidopsis and validated our method by comparing transient expression with expression in stably transformed plants. We also tested >100 synthetic parts in sorghum (Sorghum bicolor) protoplasts, and the results showed that our method works in diverse plant groups. Our approach enables the construction of tunable gene circuits in complex eukaryotic organisms.

Spontaneous Ca(2+) transients in interstitial cells of Cajal located within the deep muscular plexus of the murine small intestine.

PubMed

Baker, Salah A; Drumm, Bernard T; Saur, Dieter; Hennig, Grant W; Ward, Sean M; Sanders, Kenton M

2016-06-15

Interstitial cells of Cajal at the level of the deep muscular plexus (ICC-DMP) in the small intestine generate spontaneous Ca(2+) transients that consist of localized Ca(2+) events and limited propagating Ca(2+) waves. Ca(2+) transients in ICC-DMP display variable characteristics: from discrete, highly localized Ca(2+) transients to regionalized Ca(2+) waves with variable rates of occurrence, amplitude, duration and spatial spread. Ca(2+) transients fired stochastically, with no cellular or multicellular rhythmic activity being observed. No correlation was found between the firing sites in adjacent cells. Ca(2+) transients in ICC-DMP are suppressed by the ongoing release of inhibitory neurotransmitter(s). Functional intracellular Ca(2+) stores are essential for spontaneous Ca(2+) transients, and the sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump is necessary for maintenance of spontaneity. Ca(2+) release mechanisms involve both ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3 Rs). Release from these channels is interdependent. ICC express transcripts of multiple RyRs and InsP3 Rs, with Itpr1 and Ryr2 subtypes displaying the highest expression. Interstitial cells of Cajal in the deep muscular plexus of the small intestine (ICC-DMP) are closely associated with varicosities of enteric motor neurons and generate responses contributing to neural regulation of intestinal motility. Responses of ICC-DMP are mediated by activation of Ca(2+) -activated Cl(-) channels; thus, Ca(2+) signalling is central to the behaviours of these cells. Confocal imaging was used to characterize the nature and mechanisms of Ca(2+) transients in ICC-DMP within intact jejunal muscles expressing a genetically encoded Ca(2+) indicator (GCaMP3) selectively in ICC. ICC-DMP displayed spontaneous Ca(2+) transients that ranged from discrete, localized events to waves that propagated over variable distances. The occurrence of Ca(2+) transients was highly variable, and it was determined that firing was stochastic in nature. Ca(2+) transients were tabulated in multiple cells within fields of view, and no correlation was found between the events in adjacent cells. TTX (1 μm) significantly increased the occurrence of Ca(2+) transients, suggesting that ICC-DMP contributes to the tonic inhibition conveyed by ongoing activity of inhibitory motor neurons. Ca(2+) transients were minimally affected after 12 min in Ca(2+) free solution, indicating these events do not depend immediately upon Ca(2+) influx. However, inhibitors of sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump and blockers of inositol triphosphate receptor (InsP3 R) and ryanodine receptor (RyR) channels blocked ICC Ca(2+) transients. These data suggest an interdependence between RyR and InsP3 R in the generation of Ca(2+) transients. Itpr1 and Ryr2 were the dominant transcripts expressed by ICC. These findings provide the first high-resolution recording of the subcellular Ca(2+) dynamics that control the behaviour of ICC-DMP in situ. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Spontaneous Ca2+ transients in interstitial cells of Cajal located within the deep muscular plexus of the murine small intestine

PubMed Central

Baker, Salah A.; Drumm, Bernard T.; Saur, Dieter; Hennig, Grant W.; Ward, Sean M.

2016-01-01

Key points Interstitial cells of Cajal at the level of the deep muscular plexus (ICC‐DMP) in the small intestine generate spontaneous Ca2+ transients that consist of localized Ca2+ events and limited propagating Ca2+ waves.Ca2+ transients in ICC‐DMP display variable characteristics: from discrete, highly localized Ca2+ transients to regionalized Ca2+ waves with variable rates of occurrence, amplitude, duration and spatial spread.Ca2+ transients fired stochastically, with no cellular or multicellular rhythmic activity being observed. No correlation was found between the firing sites in adjacent cells.Ca2+ transients in ICC‐DMP are suppressed by the ongoing release of inhibitory neurotransmitter(s).Functional intracellular Ca2+ stores are essential for spontaneous Ca2+ transients, and the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) pump is necessary for maintenance of spontaneity.Ca2+ release mechanisms involve both ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3Rs). Release from these channels is interdependent.ICC express transcripts of multiple RyRs and InsP3Rs, with Itpr1 and Ryr2 subtypes displaying the highest expression. Abstract Interstitial cells of Cajal in the deep muscular plexus of the small intestine (ICC‐DMP) are closely associated with varicosities of enteric motor neurons and generate responses contributing to neural regulation of intestinal motility. Responses of ICC‐DMP are mediated by activation of Ca2+‐activated Cl− channels; thus, Ca2+ signalling is central to the behaviours of these cells. Confocal imaging was used to characterize the nature and mechanisms of Ca2+ transients in ICC‐DMP within intact jejunal muscles expressing a genetically encoded Ca2+ indicator (GCaMP3) selectively in ICC. ICC‐DMP displayed spontaneous Ca2+ transients that ranged from discrete, localized events to waves that propagated over variable distances. The occurrence of Ca2+ transients was highly variable, and it was determined that firing was stochastic in nature. Ca2+ transients were tabulated in multiple cells within fields of view, and no correlation was found between the events in adjacent cells. TTX (1 μm) significantly increased the occurrence of Ca2+ transients, suggesting that ICC‐DMP contributes to the tonic inhibition conveyed by ongoing activity of inhibitory motor neurons. Ca2+ transients were minimally affected after 12 min in Ca2+ free solution, indicating these events do not depend immediately upon Ca2+ influx. However, inhibitors of sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) pump and blockers of inositol triphosphate receptor (InsP3R) and ryanodine receptor (RyR) channels blocked ICC Ca2+ transients. These data suggest an interdependence between RyR and InsP3R in the generation of Ca2+ transients. Itpr1 and Ryr2 were the dominant transcripts expressed by ICC. These findings provide the first high‐resolution recording of the subcellular Ca2+ dynamics that control the behaviour of ICC‐DMP in situ. PMID:26824875

LPJ-GUESS Simulated North America Vegetation for 21-0 ka Using the TraCE-21ka Climate Simulation

NASA Astrophysics Data System (ADS)

Shafer, S. L.; Bartlein, P. J.

2016-12-01

Transient climate simulations that span multiple millennia (e.g., TraCE-21ka) have become more common as computing power has increased, allowing climate models to complete long simulations in relatively short periods of time (i.e., months). These climate simulations provide information on the potential rate, variability, and spatial expression of past climate changes. They also can be used as input data for other environmental models to simulate transient changes for different components of paleoenvironmental systems, such as vegetation. Long, transient paleovegetation simulations can provide information on a range of ecological processes, describe the spatial and temporal patterns of changes in species distributions, and identify the potential locations of past species refugia. Paleovegetation simulations also can be used to fill in spatial and temporal gaps in observed paleovegetation data (e.g., pollen records from lake sediments) and to test hypotheses of past vegetation change. We used the TraCE-21ka transient climate simulation for 21-0 ka from CCSM3, a coupled atmosphere-ocean general circulation model. The TraCE-21ka simulated temperature, precipitation, and cloud data were regridded onto a 10-minute grid of North America. These regridded climate data, along with soil data and atmospheric carbon dioxide concentrations, were used as input to LPJ-GUESS, a general ecosystem model, to simulate North America vegetation from 21-0 ka. LPJ-GUESS simulates many of the processes controlling the distribution of vegetation (e.g., competition), although some important processes (e.g., dispersal) are not simulated. We evaluate the LPJ-GUESS-simulated vegetation (in the form of plant functional types and biomes) for key time periods and compare the simulated vegetation with observed paleovegetation data, such as data archived in the Neotoma Paleoecology Database. In general, vegetation simulated by LPJ-GUESS reproduces the major North America vegetation patterns (e.g., forest, grassland) with regional areas of disagreement between simulated and observed vegetation. We describe the regions and time periods with the greatest data-model agreement and disagreement, and discuss some of the strengths and weaknesses of both the simulated climate and simulated vegetation data.

Detection of phasic dopamine by D1 and D2 striatal medium spiny neurons.

PubMed

Yapo, Cedric; Nair, Anu G; Clement, Lorna; Castro, Liliana R; Hellgren Kotaleski, Jeanette; Vincent, Pierre

2017-12-15

Brief dopamine events are critical actors of reward-mediated learning in the striatum; the intracellular cAMP-protein kinase A (PKA) response of striatal medium spiny neurons to such events was studied dynamically using a combination of biosensor imaging in mouse brain slices and in silico simulations. Both D1 and D2 medium spiny neurons can sense brief dopamine transients in the sub-micromolar range. While dopamine transients profoundly change cAMP levels in both types of medium spiny neurons, the PKA-dependent phosphorylation level remains unaffected in D2 neurons. At the level of PKA-dependent phosphorylation, D2 unresponsiveness depends on protein phosphatase-1 (PP1) inhibition by DARPP-32. Simulations suggest that D2 medium spiny neurons could detect transient dips in dopamine level. The phasic release of dopamine in the striatum determines various aspects of reward and action selection, but the dynamics of the dopamine effect on intracellular signalling remains poorly understood. We used genetically encoded FRET biosensors in striatal brain slices to quantify the effect of transient dopamine on cAMP or PKA-dependent phosphorylation levels, and computational modelling to further explore the dynamics of this signalling pathway. Medium-sized spiny neurons (MSNs), which express either D 1 or D 2 dopamine receptors, responded to dopamine by an increase or a decrease in cAMP, respectively. Transient dopamine showed similar sub-micromolar efficacies on cAMP in both D1 and D2 MSNs, thus challenging the commonly accepted notion that dopamine efficacy is much higher on D 2 than on D 1 receptors. However, in D2 MSNs, the large decrease in cAMP level triggered by transient dopamine did not translate to a decrease in PKA-dependent phosphorylation level, owing to the efficient inhibition of protein phosphatase 1 by DARPP-32. Simulations further suggested that D2 MSNs can also operate in a 'tone-sensing' mode, allowing them to detect transient dips in basal dopamine. Overall, our results show that D2 MSNs may sense much more complex patterns of dopamine than previously thought. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Mapping Rotational Wavepacket Dynamics with Chirped Probe Pulses

NASA Astrophysics Data System (ADS)

Romanov, Dmitri; Odhner, Johanan; Levis, Robert

2014-05-01

We develop an analytical model description of the strong-field pump-probe polarization spectroscopy of rotational transients in molecular gases in a situation when the probe pulse is considerably chirped: the frequency modulation over the pulse duration is comparable with the carrier frequency. In this scenario, a femtosecond pump laser pulse prepares a rotational wavepacket in a gas-phase sample at room temperature. The rotational revivals of the wavepacket are then mapped onto a chirped broadband probe pulse derived from a laser filament. The slow-varying envelope approximation being inapplicable, an alternative approach is proposed which is capable of incorporating the substantial chirp and the related temporal dispersion of refractive indices. Analytical expressions are obtained for the probe signal modulation over the interaction region and for the resulting heterodyned transient birefringence spectra. Dependencies of the outputs on the probe pulse parameters reveal the trade-offs and the ways to optimize the temporal-spectral imaging. The results are in good agreement with the experiments on snapshot imaging of rotational revival patterns in nitrogen gas. We gratefully acknowledge financial support through AFOSR MURI Grant No. FA9550-10-1-0561.

Characterization of Transient Receptor Potential Vanilloid-1 (TRPV1) Variant Activation by Coal Fly Ash Particles and Associations with Altered Transient Receptor Potential Ankyrin-1 (TRPA1) Expression and Asthma*

PubMed Central

Stockmann, Chris; Romero, Erin G.; Lu, Zhenyu; Shapiro, Darien; Stone, Bryan L.; Fassl, Bernhard; Nkoy, Flory; Uchida, Derek A.; Ward, Robert M.; Veranth, John M.; Reilly, Christopher A.

2016-01-01

Transient receptor potential (TRP) channels are activated by environmental particulate materials. We hypothesized that polymorphic variants of transient receptor potential vanilloid-1 (TRPV1) would be uniquely responsive to insoluble coal fly ash compared with the prototypical soluble agonist capsaicin. Furthermore, these changes would manifest as differences in lung cell responses to these agonists and perhaps correlate with changes in asthma symptom control. The TRPV1-I315M and -T469I variants were more responsive to capsaicin and coal fly ash. The I585V variant was less responsive to coal fly ash particles due to reduced translation of protein and an apparent role for Ile-585 in activation by particles. In HEK-293 cells, I585V had an inhibitory effect on wild-type TRPV1 expression, activation, and internalization/agonist-induced desensitization. In normal human bronchial epithelial cells, IL-8 secretion in response to coal fly ash treatment was reduced for cells heterozygous for TRPV1-I585V. Finally, both the I315M and I585V variants were associated with worse asthma symptom control with the effects of I315M manifesting in mild asthma and those of the I585V variant manifesting in severe, steroid-insensitive individuals. This effect may be due in part to increased transient receptor potential ankyrin-1 (TRPA1) expression by lung epithelial cells expressing the TRPV1-I585V variant. These findings suggest that specific molecular interactions control TRPV1 activation by particles, differential activation, and desensitization of TRPV1 by particles and/or other agonists, and cellular changes in the expression of TRPA1 as a result of I585V expression could contribute to variations in asthma symptom control. PMID:27758864

Characterization of Transient Receptor Potential Vanilloid-1 (TRPV1) Variant Activation by Coal Fly Ash Particles and Associations with Altered Transient Receptor Potential Ankyrin-1 (TRPA1) Expression and Asthma.

PubMed

Deering-Rice, Cassandra E; Stockmann, Chris; Romero, Erin G; Lu, Zhenyu; Shapiro, Darien; Stone, Bryan L; Fassl, Bernhard; Nkoy, Flory; Uchida, Derek A; Ward, Robert M; Veranth, John M; Reilly, Christopher A

2016-11-25

Transient receptor potential (TRP) channels are activated by environmental particulate materials. We hypothesized that polymorphic variants of transient receptor potential vanilloid-1 (TRPV1) would be uniquely responsive to insoluble coal fly ash compared with the prototypical soluble agonist capsaicin. Furthermore, these changes would manifest as differences in lung cell responses to these agonists and perhaps correlate with changes in asthma symptom control. The TRPV1-I315M and -T469I variants were more responsive to capsaicin and coal fly ash. The I585V variant was less responsive to coal fly ash particles due to reduced translation of protein and an apparent role for Ile-585 in activation by particles. In HEK-293 cells, I585V had an inhibitory effect on wild-type TRPV1 expression, activation, and internalization/agonist-induced desensitization. In normal human bronchial epithelial cells, IL-8 secretion in response to coal fly ash treatment was reduced for cells heterozygous for TRPV1-I585V. Finally, both the I315M and I585V variants were associated with worse asthma symptom control with the effects of I315M manifesting in mild asthma and those of the I585V variant manifesting in severe, steroid-insensitive individuals. This effect may be due in part to increased transient receptor potential ankyrin-1 (TRPA1) expression by lung epithelial cells expressing the TRPV1-I585V variant. These findings suggest that specific molecular interactions control TRPV1 activation by particles, differential activation, and desensitization of TRPV1 by particles and/or other agonists, and cellular changes in the expression of TRPA1 as a result of I585V expression could contribute to variations in asthma symptom control. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The role of transient receptor potential vanilloid type-2 ion channels in innate and adaptive immune responses

PubMed Central

Santoni, Giorgio; Farfariello, Valerio; Liberati, Sonia; Morelli, Maria B.; Nabissi, Massimo; Santoni, Matteo; Amantini, Consuelo

2013-01-01

The transient receptor potential vanilloid type-2 (TRPV2), belonging to the transient receptor potential channel family, is a specialized ion channel expressed in human and other mammalian immune cells. This channel has been found to be expressed in CD34+ hematopoietic stem cells, where its cytosolic Ca2+ activity is crucial for stem/progenitor cell cycle progression, growth, and differentiation. In innate immune cells, TRPV2 is expressed in granulocytes, macrophages, and monocytes where it stimulates fMet-Leu-Phe migration, zymosan-, immunoglobulin G-, and complement-mediated phagocytosis, and lipopolysaccharide-induced tumor necrosis factor-alpha and interleukin-6 production. In mast cells, activation of TRPV2 allows intracellular Ca2+ ions flux, thus stimulating protein kinase A-dependent degranulation. In addition, TRPV2 is highly expressed in CD56+ natural killer cells. TRPV2 orchestrates Ca2+ signal in T cell activation, proliferation, and effector functions. Moreover, messenger RNA for TRPV2 are expressed in CD4+ and CD8+ T lymphocytes. Finally, TRPV2 is expressed in CD19+ B lymphocytes where it regulates Ca2+ release during B cell development and activation. Overall, the specific expression of TRPV2 in immune cells suggests a role in immune-mediated diseases and offers new potential targets for immunomodulation. PMID:23420671

Temporal evolution of age data under transient pumping conditions

NASA Astrophysics Data System (ADS)

Leray, S.; De Dreuzy, J.; Aquilina, L.; Vergnaud, V.; Labasque, T.; Bour, O.; Le Borgne, T.

2013-12-01

While most age data derived from tracers have been analyzed in steady-state flow conditions, we determine their temporal evolution under transient pumping conditions. Starting pumping in a well modifies the natural flow patterns induced by the topographical gradient to a mainly convergent flow to the well. Our study is based on a set of models made up of a shallowly dipping aquifer overlain by a less permeable aquitard. These settings are characteristic of the crystalline aquifer of Plœmeur (Brittany, France) located in a highly fractured zone at the contact between a granite and micaschists. Under a pseudo steady-state flow assumption (instantaneous shift between two steady-state flow fields), we solve the transport equation with a backward particle-tracking method and determine the temporal evolution of the concentrations at the pumping well of the four atmospheric tracers CFC 11, CFC 12, CFC 113 and SF6. We show that apparent ages deduced from these concentrations evolve both because of the flow patterns modifications and because of the non-linear evolution of the atmospheric tracer concentrations. Flow patterns modifications only intervene just after the start of pumping, when the initially piston-like residence time distribution is transformed to a broader distribution mixing residence times from a wide variety of flow lines. Later, while flow patterns and the supplying volume of the pumping well still evolve, the residence time distributions are hardly modified and apparent ages are solely altered by the non-linear atmospheric tracer concentrations that progressively modifies the weighting of the residence time distribution. These results are confirmed by the observations at the site of Plœmeur in the pumping area. First, long term chloride observations confirm the quick evolution of the flow patterns after the start of pumping. Second, posterior and more recent evolutions of apparent ages derived from CFCs are consistent with the modeling results revealing in turn the marginal effect of the 20-year pumping on the first 70 years of the residence time distribution. We conclude that the temporal evolution of apparent ages should be used with great care for identifying the temporal evolution of the flow patterns as the apparent age evolution can have two sources - the transient flow patterns and transient tracer atmospheric concentrations. We argue that both evolutions either controlled by transient flow patterns or by transient tracer atmospheric concentrations provide key information that can be further used for the characterization of the hydrogeological system. This study illustrates that the temporal evolution of apparent ages could be used for models segregation and slightly compensate for the small number of tracers.

Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.

PubMed

Bleckmann, Maren; Fritz, Markus H-Y; Bhuju, Sabin; Jarek, Michael; Schürig, Margitta; Geffers, Robert; Benes, Vladimir; Besir, Hüseyin; van den Heuvel, Joop

2015-01-01

The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins--especially those entering the secretory pathway--provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.

Mir-17-3p Controls Spinal Neural Progenitor Patterning by Regulating Olig2/Irx3 Cross-repressive Loop

PubMed Central

Chen, Jun-An; Huang, Yuan-Ping; Mazzoni, Esteban O.; Tan, G. Christopher; Zavadil, Jiri; Wichterle, Hynek

2011-01-01

SUMMARY Neural patterning relies on transcriptional cross-repressive interactions that ensure unequivocal assignment of neural progenitor identity to proliferating cells. Progenitors of spinal motor neurons (pMN) and V2 interneurons (p2) are specified by a pair of cross-repressive transcription factors Olig2 and Irx3. Lineage tracing revealed that many p2 progenitors transiently express the pMN marker Olig2 during spinal cord development. Here we demonstrate that the repression of Olig2 in p2 domain is controlled by mir-17-3p microRNA-mediated silencing of Olig2 mRNA. Mice lacking all microRNAs or just the mir-17~92 cluster manifest a dorsal shift in pMN/p2 boundary and impairment in the production of V2 interneurons. Our findings suggest that microRNA-mediated repression of Olig2 mRNA plays a critical role during the patterning of ventral spinal progenitor domains by shifting the balance of cross-repressive interactions between Olig2 and Irx3 transcription factors. PMID:21338882

Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: The role of TRPC channels

PubMed Central

Kumar, Sanjay; Chakraborty, Saikat; Barbosa, Cindy; Brustovetsky, Tatiana; Brustovetsky, Nickolay; Obukhov, Alexander G.

2014-01-01

Transient Receptor Potential Canonical (TRPC) channels are implicated in modulating neurite outgrowth. The expression pattern of TRPC changes significantly during brain development, suggesting that fine-tuning TRPC expression may be important for orchestrating neuritogenesis. To study how alterations in the TRPC expression pattern affect neurite outgrowth, we used nerve growth factor (NGF)-differentiated rat pheochromocytoma 12 (PC12) cells, a model system for neuritogenesis. In PC12 cells, NGF markedly up-regulated TRPC1 and TRPC6 expression, but down-regulated TRPC5 expression while promoting neurite outgrowth. Overexpression of TRPC1 augmented, whereas TRPC5 overexpression decelerated NGF-induced neurite outgrowth. Conversely, shRNA-mediated knockdown of TRPC1 decreased, whereas shRNA-mediated knockdown of TRPC5 increased NGF-induced neurite extension. Endogenous TRPC1 attenuated the anti-neuritogenic effect of overexpressed TRPC5 in part by forming the heteromeric TRPC1–TRPC5 channels. Previous reports suggested that TRPC6 may facilitate neurite outgrowth. However, we found that TRPC6 overexpression slowed down neuritogenesis, whereas dominant negative TRPC6 (DN-TRPC6) facilitated neurite outgrowth in NGF-differentiated PC12 cells. Consistent with these findings, hyperforin, a neurite outgrowth promoting factor, decreased TRPC6 expression in NGF-differentiated PC12 cells. Using pharmacological and molecular biological approaches, we determined that NGF up-regulated TRPC1 and TRPC6 expression via a p75NTR-IKK2-dependent pathway that did not involve TrkA receptor signaling in PC12 cells. Similarly, NGF up-regulated TRPC1 and TRPC6 via an IKK2 dependent pathway in primary cultured hippocampal neurons. Thus, our data suggest that a balance of TRPC1, TRPC5, and TRPC6 expression determines neurite extension rate in neural cells, with TRPC6 emerging as an NGF-dependent “molecular damper” maintaining a submaximal velocity of neurite extension. PMID:21618530

Systematic Onset of Periodic Patterns in Random Disk Packings

NASA Astrophysics Data System (ADS)

Topic, Nikola; Pöschel, Thorsten; Gallas, Jason A. C.

2018-04-01

We report evidence of a surprising systematic onset of periodic patterns in very tall piles of disks deposited randomly between rigid walls. Independently of the pile width, periodic structures are always observed in monodisperse deposits containing up to 1 07 disks. The probability density function of the lengths of disordered transient phases that precede the onset of periodicity displays an approximately exponential tail. These disordered transients may become very large when the channel width grows without bound. For narrow channels, the probability density of finding periodic patterns of a given period displays a series of discrete peaks, which, however, are washed out completely when the channel width grows.

Integrity of Narrow Epithelial Tubes in the C. elegans Excretory System Requires a Transient Luminal Matrix

PubMed Central

Ayala-Figueroa, Jesus; Parry, Jean M.; Pu, Pu; Hall, David H.

2016-01-01

Most epithelial cells secrete a glycoprotein-rich apical extracellular matrix that can have diverse but still poorly understood roles in development and physiology. Zona Pellucida (ZP) domain glycoproteins are common constituents of these matrices, and their loss in humans is associated with a number of diseases. Understanding of the functions, organization and regulation of apical matrices has been hampered by difficulties in imaging them both in vivo and ex vivo. We identified the PAN-Apple, mucin and ZP domain glycoprotein LET-653 as an early and transient apical matrix component that shapes developing epithelia in C. elegans. LET-653 has modest effects on shaping of the vulva and epidermis, but is essential to prevent lumen fragmentation in the very narrow, unicellular excretory duct tube. We were able to image the transient LET-653 matrix by both live confocal imaging and transmission electron microscopy. Structure/function and fluorescence recovery after photobleaching studies revealed that LET-653 exists in two separate luminal matrix pools, a loose fibrillar matrix in the central core of the lumen, to which it binds dynamically via its PAN domains, and an apical-membrane-associated matrix, to which it binds stably via its ZP domain. The PAN domains are both necessary and sufficient to confer a cyclic pattern of duct lumen localization that precedes each molt, while the ZP domain is required for lumen integrity. Ectopic expression of full-length LET-653, but not the PAN domains alone, could expand lumen diameter in the developing gut tube, where LET-653 is not normally expressed. Together, these data support a model in which the PAN domains regulate the ability of the LET-653 ZP domain to interact with other factors at the apical membrane, and this ZP domain interaction promotes expansion and maintenance of lumen diameter. These data identify a transient apical matrix component present prior to cuticle secretion in C. elegans, demonstrate critical roles for this matrix component in supporting lumen integrity within narrow bore tubes such as those found in the mammalian microvasculature, and reveal functional importance of the evolutionarily conserved ZP domain in this tube protecting activity. PMID:27482894

Retrovirus-based vectors for transient and permanent cell modification.

PubMed

Schott, Juliane W; Hoffmann, Dirk; Schambach, Axel

2015-10-01

Retroviral vectors are commonly employed for long-term transgene expression via integrating vector technology. However, three alternative retrovirus-based platforms are currently available that allow transient cell modification. Gene expression can be mediated from either episomal DNA or RNA templates, or selected proteins can be directly transferred through retroviral nanoparticles. The different technologies are functionally graded with respect to safety, expression magnitude and expression duration. Improvement of the initial technologies, including modification of vector designs, targeted increase in expression strength and duration as well as improved safety characteristics, has allowed maturation of retroviral systems into efficient and promising tools that meet the technological demands of a wide variety of potential application areas. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hox genes, digit identities and the theropod/bird transition.

PubMed

Galis, Frietson; Kundrát, Martin; Metz, Johan A J

2005-05-15

Vargas and Fallon (2005. J Exp Zool (Mol Dev Evol) 304B:86-90) propose that Hox gene expression patterns indicate that the most anterior digit in bird wings is homologous to digit 1 rather than to digit 2 in other amniotes. This interpretation is based on the presence of Hoxd13 expression in combination with the absence of Hoxd12 expression in the second digit condensation from which this digit develops (the first condensation is transiently present). This is a pattern that is similar to that in the developing digit 1 of the chicken foot and the mouse hand and foot. They have tested this new hypothesis by analysing Hoxd12 and Hoxd13 expression patterns in two polydactylous chicken mutants, Silkie and talpid2. They conclude that the data support the notion that the most anterior remaining digit of the bird wing is homologous to digit 1 in other amniotes either in a standard phylogenetic sense, or alternatively in a (limited) developmental sense in agreement with the Frameshift Hypothesis of Wagner and Gautier (1999, i.e., that the developmental pathway is homologous to the one that leads to a digit 1 identity in other amniotes, although it occurs in the second instead of the first digit condensation). We argue that the Hoxd12 and Hoxd13 expression patterns found for these and other limb mutants do not allow distinguishing between the hypothesis of Vargas and Fallon (2005. J Exp Zool (Mol Dev Evol) 304B:86-90) and the alternative one, i.e., the most anterior digit in bird wings is homologous to digit 2 in other amniotes, in a phylogenetic or developmental sense. Therefore, at the moment the data on limb mutants does not present a challenge to the hypothesis, based on other developmental data (Holmgren, 1955. Acta Zool 36:243-328; Hinchliffe, 1984. In: Hecht M, Ostrom JH, Viohl G, Wellnhofer P, editors. The beginnings of birds. Eichstätt: Freunde des Jura-Museum. p 141-147; Burke and Feduccia, 1997. Science 278:666-668; Kundrát et al., 2002. J Exp Zool (Mol Dev Evol) 294B:151-159; Larsson and Wagner, 2002. J Exp Zool (Mol Dev Evol) 294B:146-151; Feduccia and Nowicki, 2002. Naturwissenschaften 89:391-393), that the digits of bird wings are homologous to digits 2,3,4 in amniotes. We recommend further testing of the hypothesis by comparing Hoxd expression patterns in different taxa. Copyright 2005 Wiley-Liss, Inc

Lag Phase Is a Distinct Growth Phase That Prepares Bacteria for Exponential Growth and Involves Transient Metal Accumulation

PubMed Central

Rolfe, Matthew D.; Rice, Christopher J.; Lucchini, Sacha; Pin, Carmen; Thompson, Arthur; Cameron, Andrew D. S.; Alston, Mark; Stringer, Michael F.; Betts, Roy P.; Baranyi, József; Peck, Michael W.

2012-01-01

Lag phase represents the earliest and most poorly understood stage of the bacterial growth cycle. We developed a reproducible experimental system and conducted functional genomic and physiological analyses of a 2-h lag phase in Salmonella enterica serovar Typhimurium. Adaptation began within 4 min of inoculation into fresh LB medium with the transient expression of genes involved in phosphate uptake. The main lag-phase transcriptional program initiated at 20 min with the upregulation of 945 genes encoding processes such as transcription, translation, iron-sulfur protein assembly, nucleotide metabolism, LPS biosynthesis, and aerobic respiration. ChIP-chip revealed that RNA polymerase was not “poised” upstream of the bacterial genes that are rapidly induced at the beginning of lag phase, suggesting a mechanism that involves de novo partitioning of RNA polymerase to transcribe 522 bacterial genes within 4 min of leaving stationary phase. We used inductively coupled plasma mass spectrometry (ICP-MS) to discover that iron, calcium, and manganese are accumulated by S. Typhimurium during lag phase, while levels of cobalt, nickel, and sodium showed distinct growth-phase-specific patterns. The high concentration of iron during lag phase was associated with transient sensitivity to oxidative stress. The study of lag phase promises to identify the physiological and regulatory processes responsible for adaptation to new environments. PMID:22139505

Kupffer's vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut.

PubMed

Essner, Jeffrey J; Amack, Jeffrey D; Nyholm, Molly K; Harris, Erin B; Yost, H Joseph

2005-03-01

Monocilia have been proposed to establish the left-right (LR) body axis in vertebrate embryos by creating a directional fluid flow that triggers asymmetric gene expression. In zebrafish, dorsal forerunner cells (DFCs) express a conserved ciliary dynein gene (left-right dynein-related1, lrdr1) and form a ciliated epithelium inside a fluid-filled organ called Kupffer's vesicle (KV). Here, videomicroscopy demonstrates that cilia inside KV are motile and create a directional fluid flow just prior to the onset of asymmetric gene expression in lateral cells. Laser ablation of DFCs and surgical disruption of KV provide direct evidence that ciliated KV cells are required during early somitogenesis for subsequent LR patterning in the brain, heart and gut. Antisense morpholinos against lrdr1 disrupt KV fluid flow and perturb LR development. Furthermore, lrdr1 morpholinos targeted to DFC/KV cells demonstrate that Lrdr1 functions in these ciliated cells to control LR patterning. This provides the first direct evidence, in any vertebrate, that impairing cilia function in derivatives of the dorsal organizer, and not in other cells that express ciliogenic genes, alters LR development. Finally, genetic analysis reveals novel roles for the T-box transcription factor no tail and the Nodal signaling pathway as upstream regulators of lrdr1 expression and KV morphogenesis. We propose that KV is a transient embryonic 'organ of asymmetry' that directs LR development by establishing a directional fluid flow. These results suggest that cilia are an essential component of a conserved mechanism that controls the transition from bilateral symmetry to LR asymmetry in vertebrates.

Steroid signaling system responds differently to temperature and hormone manipulation in the red-eared slider turtle (Trachemys scripta elegans), a reptile with temperature-dependent sex determination.

PubMed

Ramsey, M; Crews, D

2007-01-01

Many reptiles, including the red-eared slider turtle (Trachemys scripta elegans), exhibit temperature-dependent sex determination (TSD). Temperature determines gonadal sex during the middle of embryogenesis, or the temperature-sensitive period (TSP), when gonadal sex is labile to both temperature and hormones--particularly estrogen. The biological actions of steroid hormones are mediated by their receptors as defined here as the classic transcriptional regulation of target genes. To elucidate estrogen action during sex determination, we examined estrogen receptor alpha (Esr1, hereafter referred to as ERalpha), estrogen receptor beta (Esr2, hereafter referred to as ERbeta), and androgen receptor (Ar, hereafter referred to as AR) expression in slider turtle gonads before, during and after the TSP, as well as following sex reversal via temperature or steroid hormone manipulation. ERalpha and AR levels spike at the female-producing temperature while ovarian sex is determined, but none of the receptors exhibited sexually dimorphic localization within the gonad prior to morphological differentiation. All three receptors respond differentially to sex-reversing treatments. When shifted to female-producing temperatures, embryos maintain ERalpha and AR expression while ERbeta is reduced. When shifted to male-producing temperatures, medullary expression of all three receptors is reduced. Feminization via estradiol (E(2)) treatment at a male-producing temperature profoundly changed the expression patterns for all three receptors. ERalpha and ERbeta redirected to the cortex in E(2)-created ovaries, while AR medullary expression was transiently reduced. Although warmer incubation temperature and estrogen result in the same endpoint (ovarian development), our results indicate different steroid signaling patterns between temperature- and estrogen-induced feminization. 2007 S. Karger AG, Basel

Expression Patterns Conferred by Tyrosine/Dihydroxyphenylalanine Decarboxylase Promoters from Opium Poppy Are Conserved in Transgenic Tobacco1

PubMed Central

Facchini, Peter J.; Penzes-Yost, Catherine; Samanani, Nailish; Kowalchuk, Brett

1998-01-01

Opium poppy (Papaver somniferum) contains a large family of tyrosine/dihydroxyphenylalanine decarboxylase (tydc) genes involved in the biosynthesis of benzylisoquinoline alkaloids and cell wall-bound hydroxycinnamic acid amides. Eight members from two distinct gene subfamilies have been isolated, tydc1, tydc4, tydc6, tydc8, and tydc9 in one group and tydc2, tydc3, and tydc7 in the other. The tydc8 and tydc9 genes were located 3.2 kb apart on one genomic clone, suggesting that the family is clustered. Transcripts for most tydc genes were detected only in roots. Only tydc2 and tydc7 revealed expression in both roots and shoots, and TYDC3 mRNAs were the only specific transcripts detected in seedlings. TYDC1, TYDC8, and TYDC9 mRNAs, which occurred in roots, were not detected in elicitor-treated opium poppy cultures. Expression of tydc4, which contains a premature termination codon, was not detected under any conditions. Five tydc promoters were fused to the β-glucuronidase (GUS) reporter gene in a binary vector. All constructs produced transient GUS activity in microprojectile-bombarded opium poppy and tobacco (Nicotiana tabacum) cell cultures. The organ- and tissue-specific expression pattern of tydc promoter-GUS fusions in transgenic tobacco was generally parallel to that of corresponding tydc genes in opium poppy. GUS expression was most abundant in the internal phloem of shoot organs and in the stele of roots. Select tydc promoter-GUS fusions were also wound induced in transgenic tobacco, suggesting that the basic mechanisms of developmental and inducible tydc regulation are conserved across plant species. PMID:9733527

17β-estradiol regulates the differentiation of cementoblasts via Notch signaling cascade

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liao, Jing; Zhou, Zeyuan; Huang, Li

Estrogen has been well recognized as a key factor in the homeostasis of bone and periodontal tissue, but the way it regulates the activities of cementoblasts, the cell population maintaining cementum has not been fully understood. In this study, we examined the expression of estrogen receptor in OCCM-30 cells and the effect of 17β-estradiol (E2) on the proliferation and differentiation of OCCM-30 cells. We found that both estrogen receptor α and β were expressed in OCCM-30 cells. E2 exerted no significant influence on the proliferation of OCCM-30 cells, but inhibited the transcription and translation of BSP and Runx2 in the early phase of osteogenicmore » induction except the BSP mRNA. Afterwards in the late phase of osteogenic induction, E2 enhanced the transcription and translation of BSP and Runx2 and promoted the calcium deposition. In addition, the expression level of Notch1, NICD and Hey1 mRNAs responded to exogenous E2 in a pattern similar to that of the osteoblastic markers. DAPT could attenuate the effect of E2 on the expression of osteoblastic markers. These findings indicated that E2 might regulate the differentiation of cementoblasts via Notch signaling. - Highlights: • 17β-estradiol showed no significant effect on the proliferation of cementoblasts. • 17β-estradiol promoted the osteoblastic differentiation of cementoblasts despite of an early transient inhibition. • Notch signaling was regulated by 17β-estradiol and was responsible for mediating the effect of E2 on cementoblasts. • Hey1 might display an opposite expression pattern to Notch signaling in certain circumstances.« less

Phylogenetic analysis of cnidarian peroxiredoxins and stress-responsive expression in the estuarine sea anemone Nematostella vectensis.

PubMed

Helm, Rebecca R; Martín-Díaz, Maria Laura; Tarrant, Ann M

2018-07-01

Peroxiredoxins (PRXs) are a family of antioxidant enzymes present in all domains of life. To date, the diversity and function of peroxiredoxins within animals have only been studied in a few model species. Thus, we sought to characterize peroxiredoxin diversity in cnidarians and to gain insight into their function in one cnidarian-the sea anemone Nematostella vectensis. Phylogenetic analysis using all six known PRX subfamilies (PRX1-4, PRX5, PRX6, PRXQ/AHPE1, TPX, BCP-PRXQ) revealed that like bilaterians, cnidarians contain representatives from three subfamilies (PRX1-4, PRX5, PRX6). Within the PRX1-4 subfamily, cnidarian sequences fall into two clades: PRX4, and a cnidarian-specific clade, which we term CNID-PRX. This phylogenetic analysis demonstrates that the three PRX subfamilies present in Bilateria were also present in the last common ancestor of the Cnidaria and Bilateria, and further that diversification of the PRX1-4 subfamily has occurred within the cnidarian lineage. We next examined the impact of decreased salinity, increased temperature, and peroxide exposure on the expression of four prx genes in N. vectensis (cnid-prx, prx4, prx5, and prx6). These genes exhibited unique expression patterns in response to these environmental stressors. Expression of prx4 decreased with initial exposure to elevated temperature, cnid-prx increased with exposure to elevated temperatures as well as with hydrogen peroxide exposure, and expression of all prxs transiently decreased with reduced salinity. Predicted subcellular localization patterns also varied among PRX proteins. Together these results provide evidence that peroxiredoxins in N. vectensis serve distinct physiological roles and lay a groundwork for understanding how peroxiredoxins mediate cnidarian developmental processes and environmental responses. Copyright © 2018 Elsevier Inc. All rights reserved.

Searching for non-transposable targets of planarian nuclear PIWI in pluripotent stem cells and differentiated cells.

PubMed

Kashima, Makoto; Agata, Kiyokazu; Shibata, Norito

2018-06-01

Nuclear PIWIs together with their guide RNAs (piRNAs) epigenetically silence various genes including transposons in many organisms. In planarians, the nuclear piwi family gene, DjpiwiB is specifically transcribed in adult pluripotent stem cells (adult PSC, neoblast), but not in differentiated cells. However, the protein accumulates in the nuclei of both neoblasts and their descendant differentiated cells. Interestingly, PIWI(DjPiwiB)-piRNA complexes are indispensable for the repression of transposable genes at the onset of differentiation from neoblasts. Here, we conducted a comparative transcriptome analysis between control and DjpiwiB(RNAi) animals to identify non-transposable target genes of the DjPiwiB-piRNA complexes. Using bioinformatic analyses and RNAi we demonstrate that DjPiwiB-piRNA complexes are required for the proper expression of Djmcm2 and Djhistone h4 in neoblasts and that DjPiwiB-piRNA complexes regulate the transient expression of Djcalu during neoblast differentiation. Thus, DjPiwiB-piRNA complexes regulate the correct expression patterns during neoblast self-renewal and differentiation. © 2018 Japanese Society of Developmental Biologists.

Early development of the Drosophila brain: V. Pattern of postembryonic neuronal lineages expressing DE-cadherin.

PubMed

Dumstrei, Karin; Wang, Fay; Nassif, Claude; Hartenstein, Volker

2003-01-20

The Drosophila E-cadherin homolog, DE-cadherin, is expressed postembryonically by brain neuroblasts and their lineages of neurons ("secondary lineages"). DE-cadherin appears in neuroblasts as soon as they can be identified by their increase in size and then remains expressed uninterruptedly throughout larval life. DE-cadherin remains transiently expressed in the cell bodies and axons of neurons produced by neuroblast proliferation. In general, axons of neurons belonging to one lineage form tight bundles. The trajectories of these bundles are correlated with the location of the neuronal lineages to which they belong. Thus, axon bundles of lineages that are neighbors in the cortex travel parallel to each other and reach the neuropile at similar positions. It is, therefore, possible to assign coherent groups of neuroblasts and their lineages to the individual neuropile compartments and long axon tracts introduced in the accompanying articles (Nassif et al. [2003] J Comp Neurol 455:417-434; Younossi-Hartenstein et al. [2003] J Comp Neurol 455:435-450). In this study, we have reconstructed the pattern of secondary lineages and their projection in relationship to the compartments and Fasciclin II-positive long axon tracts. Based on topology and axonal trajectory, the lineages of the central brain can be subdivided into 11 groups that can be followed throughout successive larval stages. The map of larval lineages and their axonal projection will be important for future studies on postembryonic neurogenesis in Drosophila. It also lays a groundwork for investigating the role of DE-cadherin in larval brain development. Copyright 2002 Wiley-Liss, Inc.

Identification of a progenitor cell population destined to form fracture fibrocartilage callus in Dickkopf-related protein 3-green fluorescent protein reporter mice.

PubMed

Mori, Yu; Adams, Douglas; Hagiwara, Yusuke; Yoshida, Ryu; Kamimura, Masayuki; Itoi, Eiji; Rowe, David W

2016-11-01

Fracture healing is a complex biological process involving the proliferation of mesenchymal progenitor cells, and chondrogenic, osteogenic, and angiogenic differentiation. The mechanisms underlying the proliferation and differentiation of mesenchymal progenitor cells remain unclear. Here, we demonstrate Dickkopf-related protein 3 (Dkk3) expression in periosteal cells using Dkk3-green fluorescent protein reporter mice. We found that proliferation of mesenchymal progenitor cells began in the periosteum, involving Dkk3-positive cell proliferation near the fracture site. In addition, Dkk3 was expressed in fibrocartilage cells together with smooth muscle α-actin and Col3.6 in the early phase of fracture healing as a cell marker of fibrocartilage cells. Dkk3 was not expressed in mature chondrogenic cells or osteogenic cells. Transient expression of Dkk3 disappeared in the late phase of fracture healing, except in the superficial periosteal area of fracture callus. The Dkk3 expression pattern differed in newly formed type IV collagen positive blood vessels and the related avascular tissue. This is the first report that shows Dkk3 expression in the periosteum at a resting state and in fibrocartilage cells during the fracture healing process, which was associated with smooth muscle α-actin and Col3.6 expression in mesenchymal progenitor cells. These fluorescent mesenchymal lineage cells may be useful for future studies to better understand fracture healing.

Differentially Expressed microRNAs and Target Genes Associated with Plastic Internode Elongation in Alternanthera philoxeroides in Contrasting Hydrological Habitats

PubMed Central

Li, Gengyun; Deng, Ying; Geng, Yupeng; Zhou, Chengchuan; Wang, Yuguo; Zhang, Wenju; Song, Zhiping; Gao, Lexuan; Yang, Ji

2017-01-01

Phenotypic plasticity is crucial for plants to survive in changing environments. Discovering microRNAs, identifying their targets and further inferring microRNA functions in mediating plastic developmental responses to environmental changes have been a critical strategy for understanding the underlying molecular mechanisms of phenotypic plasticity. In this study, the dynamic expression patterns of microRNAs under contrasting hydrological habitats in the amphibious species Alternanthera philoxeroides were identified by time course expression profiling using high-throughput sequencing technology. A total of 128 known and 18 novel microRNAs were found to be differentially expressed under contrasting hydrological habitats. The microRNA:mRNA pairs potentially associated with plastic internode elongation were identified by integrative analysis of microRNA and mRNA expression profiles, and were validated by qRT-PCR and 5′ RLM-RACE. The results showed that both the universal microRNAs conserved across different plants and the unique microRNAs novelly identified in A. philoxeroides were involved in the responses to varied water regimes. The results also showed that most of the differentially expressed microRNAs were transiently up-/down-regulated at certain time points during the treatments. The fine-scale temporal changes in microRNA expression highlighted the importance of time-series sampling in identifying stress-responsive microRNAs and analyzing their role in stress response/tolerance. PMID:29259617

Dynamics of immediate early gene and neuropeptide gene response to prolonged immobilization stress: evidence against a critical role of the termination of exposure to the stressor.

PubMed

Trnecková, Lenka; Rotllant, David; Klenerová, Vera; Hynie, Sixtus; Armario, Antonio

2007-02-01

Stress-induced expression of immediate early genes (IEGs) appears to be transient even if the exposure to the stressor persists. However, there are some exceptions which suggest that particular characteristics of stressors can affect the dynamics of IEG expression. We studied in selected telencephalic, diencephalic and brainstem regions the mRNA levels of two clearly distinct IEGs (c-fos and arc) during prolonged exposure to a severe stressor such as immobilization (IMO) and after releasing the rats from the situation. Although regional differences were observed with the two IEGs, overall, c-fos mRNA levels progressively declined over the course of 4 h of continuous exposure to IMO, whereas arc mRNA levels were maintained at high levels in the brain regions that express this gene under stress (telencephalon). Levels of CRF hnRNA in the hypothalamus paraventricular nucleus only slightly declined during prolonged exposure to IMO. Surprisingly, termination of exposure to IMO did not modify CRF gene expression in the paraventricular nucleus or the pattern of IEGs expression, with the exception of c-fos in the lateral septum. Thus, putative signals associated to the termination of exposure to IMO were unable to modify either IEG expression in most brain areas or CRF gene expression in the paraventricular nucleus.

Terminal Transient Phase of Chaotic Transients

NASA Astrophysics Data System (ADS)

Lilienkamp, Thomas; Parlitz, Ulrich

2018-03-01

Transient chaos in spatially extended systems can be characterized by the length of the transient phase, which typically grows quickly with the system size (supertransients). For a large class of these systems, the chaotic phase terminates abruptly, without any obvious precursors in commonly used observables. Here we investigate transient spatiotemporal chaos in two different models of this class. By probing the state space using perturbed trajectories we show the existence of a "terminal transient phase," which occurs prior to the abrupt collapse of chaotic dynamics. During this phase the impact of perturbations is significantly different from the earlier transient and particular patterns of (non)susceptible regions in state space occur close to the chaotic trajectories. We therefore hypothesize that even without perturbations proper precursors for the collapse of chaotic transients exist, which might be highly relevant for coping with spatiotemporal chaos in cardiac arrhythmias or brain functionality, for example.

Depression of voltage-activated Ca2+ release in skeletal muscle by activation of a voltage-sensing phosphatase

PubMed Central

Berthier, Christine; Kutchukian, Candice; Bouvard, Clément; Okamura, Yasushi

2015-01-01

Phosphoinositides act as signaling molecules in numerous cellular transduction processes, and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) regulates the function of several types of plasma membrane ion channels. We investigated the potential role of PtdIns(4,5)P2 in Ca2+ homeostasis and excitation–contraction (E-C) coupling of mouse muscle fibers using in vivo expression of the voltage-sensing phosphatases (VSPs) Ciona intestinalis VSP (Ci-VSP) or Danio rerio VSP (Dr-VSP). Confocal images of enhanced green fluorescent protein–tagged Dr-VSP revealed a banded pattern consistent with VSP localization within the transverse tubule membrane. Rhod-2 Ca2+ transients generated by 0.5-s-long voltage-clamp depolarizing pulses sufficient to elicit Ca2+ release from the sarcoplasmic reticulum (SR) but below the range at which VSPs are activated were unaffected by the presence of the VSPs. However, in Ci-VSP–expressing fibers challenged by 5-s-long depolarizing pulses, the Ca2+ level late in the pulse (3 s after initiation) was significantly lower at 120 mV than at 20 mV. Furthermore, Ci-VSP–expressing fibers showed a reversible depression of Ca2+ release during trains, with the peak Ca2+ transient being reduced by ∼30% after the application of 10 200-ms-long pulses to 100 mV. A similar depression was observed in Dr-VSP–expressing fibers. Cav1.1 Ca2+ channel–mediated current was unaffected by Ci-VSP activation. In fibers expressing Ci-VSP and a pleckstrin homology domain fused with monomeric red fluorescent protein (PLCδ1PH-mRFP), depolarizing pulses elicited transient changes in mRFP fluorescence consistent with release of transverse tubule–bound PLCδ1PH domain into the cytosol; the voltage sensitivity of these changes was consistent with that of Ci-VSP activation, and recovery occurred with a time constant in the 10-s range. Our results indicate that the PtdIns(4,5)P2 level is tightly maintained in the transverse tubule membrane of the muscle fibers, and that VSP-induced depletion of PtdIns(4,5)P2 impairs voltage-activated Ca2+ release from the SR. Because Ca2+ release is thought to be independent from InsP3 signaling, the effect likely results from an interaction between PtdIns(4,5)P2 and a protein partner of the E-C coupling machinery. PMID:25825170

Depression of voltage-activated Ca2+ release in skeletal muscle by activation of a voltage-sensing phosphatase.

PubMed

Berthier, Christine; Kutchukian, Candice; Bouvard, Clément; Okamura, Yasushi; Jacquemond, Vincent

2015-04-01

Phosphoinositides act as signaling molecules in numerous cellular transduction processes, and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) regulates the function of several types of plasma membrane ion channels. We investigated the potential role of PtdIns(4,5)P2 in Ca(2+) homeostasis and excitation-contraction (E-C) coupling of mouse muscle fibers using in vivo expression of the voltage-sensing phosphatases (VSPs) Ciona intestinalis VSP (Ci-VSP) or Danio rerio VSP (Dr-VSP). Confocal images of enhanced green fluorescent protein-tagged Dr-VSP revealed a banded pattern consistent with VSP localization within the transverse tubule membrane. Rhod-2 Ca(2+) transients generated by 0.5-s-long voltage-clamp depolarizing pulses sufficient to elicit Ca(2+) release from the sarcoplasmic reticulum (SR) but below the range at which VSPs are activated were unaffected by the presence of the VSPs. However, in Ci-VSP-expressing fibers challenged by 5-s-long depolarizing pulses, the Ca(2+) level late in the pulse (3 s after initiation) was significantly lower at 120 mV than at 20 mV. Furthermore, Ci-VSP-expressing fibers showed a reversible depression of Ca(2+) release during trains, with the peak Ca(2+) transient being reduced by ∼30% after the application of 10 200-ms-long pulses to 100 mV. A similar depression was observed in Dr-VSP-expressing fibers. Cav1.1 Ca(2+) channel-mediated current was unaffected by Ci-VSP activation. In fibers expressing Ci-VSP and a pleckstrin homology domain fused with monomeric red fluorescent protein (PLCδ1PH-mRFP), depolarizing pulses elicited transient changes in mRFP fluorescence consistent with release of transverse tubule-bound PLCδ1PH domain into the cytosol; the voltage sensitivity of these changes was consistent with that of Ci-VSP activation, and recovery occurred with a time constant in the 10-s range. Our results indicate that the PtdIns(4,5)P2 level is tightly maintained in the transverse tubule membrane of the muscle fibers, and that VSP-induced depletion of PtdIns(4,5)P2 impairs voltage-activated Ca(2+) release from the SR. Because Ca(2+) release is thought to be independent from InsP3 signaling, the effect likely results from an interaction between PtdIns(4,5)P2 and a protein partner of the E-C coupling machinery. © 2015 Berthier et al.

Integrated cell and process engineering for improved transient production of a "difficult-to-express" fusion protein by CHO cells.

PubMed

Johari, Yusuf B; Estes, Scott D; Alves, Christina S; Sinacore, Marty S; James, David C

2015-12-01

Based on an optimized electroporation protocol, we designed a rapid, milliliter-scale diagnostic transient production assay to identify limitations in the ability of Chinese hamster ovary (CHO) cells to produce a model "difficult-to-express" homodimeric Fc-fusion protein, Sp35Fc, that exhibited very low volumetric titer and intracellular formation of disulfide-bonded oligomeric aggregates post-transfection. As expression of Sp35Fc induced an unfolded protein response in transfected host cells, we utilized the transient assay to compare, in parallel, multiple functionally diverse strategies to engineer intracellular processing of Sp35Fc in order to increase production and reduce aggregation as two discrete design objectives. Specifically, we compared the effect of (i) co-expression of ER-resident molecular chaperones (BiP, PDI, CypB) or active forms of UPR transactivators (ATF6c, XBP1s) at varying recombinant gene load, (ii) addition of small molecules known to act as chemical chaperones (PBA, DMSO, glycerol, betaine, TMAO) or modulate UPR signaling (PERK inhibitor GSK2606414) at varying concentration, (iii) a reduction in culture temperature to 32°C. Using this information, we designed a biphasic, Sp35Fc-specific transient manufacturing process mediated by lipofection that utilized CypB co-expression at an optimal Sp35Fc:CypB gene ratio of 5:1 to initially maximize transfected cell proliferation, followed by addition of a combination of PBA (0.5 mM) and glycerol (1% v/v) at the onset of stationary phase to maximize cell specific production and eliminate Sp35Fc aggregation. Using this optimal, engineered process transient Sp35Fc production was significantly increased sixfold over a 12 day production process with no evidence of disulfide-bonded aggregates. Finally, transient production in clonally derived sub-populations (derived from parental CHO host) screened for a heritably improved capability to produce Sp35Fc was also significantly improved by the optimized process, showing that protein-specific cell/process engineering can provide a solution that exceeds the limits of genetic/functional diversity within heterogeneous host cell populations. . © 2015 Wiley Periodicals, Inc.

Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells.

PubMed

De Sutter, Valerie; Vanderhaeghen, Rudy; Tilleman, Sofie; Lammertyn, Freya; Vanhoutte, Isabelle; Karimi, Mansour; Inzé, Dirk; Goossens, Alain; Hilson, Pierre

2005-12-01

Although sequence information and genome annotation are improving at an impressive pace, functional ontology is still non-existent or rudimentary for most genes. In this regard, transient expression assays are very valuable for identification of short functional segments in particular pathways, because they can be performed rapidly and at a scale unattainable in stably transformed tissues. Vectors were constructed and protocols developed for systematic transient assays in plant protoplasts. To enhance throughput and reproducibility, protoplast treatments were performed entirely by a liquid-handling robot in multiwell plates, including polyethylene glycol/Ca2+ cell transfection with plasmid mixtures, washes and lysis. All transcriptional readouts were measured using a dual firefly/Renilla luciferase assay, in which the former was controlled by a reporter promoter and the latter by the 35S CaMV promoter, which served as internal normalization standard. The automated protocols were suitable for transient assays in protoplasts prepared from cell cultures of Nicotiana tabacum Bright Yellow-2 and Arabidopsis thaliana. They were implemented in a screen to discover potential regulators of genes coding for key enzymes in nicotine biosynthesis. Two novel tobacco transcription factors were found, NtORC1 and NtJAP1, that positively regulate the putrescine N-methyltransferase (PMT) promoter. In addition, combinatorial tests showed that these two factors act synergistically to induce PMT transcriptional activity. The development and use of high-throughput plant transient expression assays are discussed.

Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits

PubMed Central

Tariq, Mohammad F.; Phillips, Ryan S.; Mosher, Bryan; Thompson, Ryan; Zhang, Ruli

2018-01-01

Abstract Transient receptor potential channel, TRPM4, the putative molecular substrate for Ca2+-activated nonselective cation current (ICAN), is hypothesized to generate bursting activity of pre-Bötzinger complex (pre-BötC) inspiratory neurons and critically contribute to respiratory rhythmogenesis. Another TRP channel, TRPC3, which mediates Na+/Ca2+ fluxes, may be involved in regulating Ca2+-related signaling, including affecting TRPM4/ICAN in respiratory pre-BötC neurons. However, TRPM4 and TRPC3 expression in pre-BötC inspiratory neurons and functional roles of these channels remain to be determined. By single-cell multiplex RT-PCR, we show mRNA expression for these channels in pre-BötC inspiratory neurons in rhythmically active medullary in vitro slices from neonatal rats and mice. Functional contributions were analyzed with pharmacological inhibitors of TRPM4 or TRPC3 in vitro as well as in mature rodent arterially perfused in situ brainstem–spinal cord preparations. Perturbations of respiratory circuit activity were also compared with those by a blocker of ICAN. Pharmacologically attenuating endogenous activation of TRPM4, TRPC3, or ICAN in vitro similarly reduced the amplitude of inspiratory motoneuronal activity without significant perturbations of inspiratory frequency or variability of the rhythm. Amplitude perturbations were correlated with reduced inspiratory glutamatergic pre-BötC neuronal activity, monitored by multicellular dynamic calcium imaging in vitro. In more intact circuits in situ, the reduction of pre-BötC and motoneuronal inspiratory activity amplitude was accompanied by reduced post-inspiratory motoneuronal activity, without disruption of rhythm generation. We conclude that endogenously activated TRPM4, which likely mediates ICAN, and TRPC3 channels in pre-BötC inspiratory neurons play fundamental roles in respiratory pattern formation but are not critically involved in respiratory rhythm generation. PMID:29435486

Transient co-expression of post-transcriptional gene silencing suppressors for increased in planta expression of a recombinant anthrax receptor fusion protein.

PubMed

Arzola, Lucas; Chen, Junxing; Rattanaporn, Kittipong; Maclean, James M; McDonald, Karen A

2011-01-01

Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin), CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA) domain of human capillary morphogenesis 2 (CMG2), an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG). We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc with nine different viral suppressors of post-transcriptional gene silencing (PTGS): p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI), with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI.

Influence of transient flooding on methane fluxes from subtropical pastures

USDA-ARS?s Scientific Manuscript database

Seasonally flooded subtropical pastures are major methane (CH4) sources, where transient flooding drives episodic and high-magnitude emissions from the underlying landscape. Understanding the mechanisms that drive these patterns is needed to better understand pasture CH4 emissions and their response...

Uridine 5′-Triphosphate Promotes In Vitro Schwannoma Cell Migration through Matrix Metalloproteinase-2 Activation

PubMed Central

Martiañez, Tania; Segura, Mònica; Figueiro-Silva, Joana; Grijota-Martinez, Carmen; Trullas, Ramón; Casals, Núria

2014-01-01

In response to peripheral nerve injury, Schwann cells adopt a migratory phenotype and modify the extracellular matrix to make it permissive for cell migration and axonal re-growth. Uridine 5′-triphosphate (UTP) and other nucleotides are released during nerve injury and activate purinergic receptors expressed on the Schwann cell surface, but little is known about the involvement of purine signalling in wound healing. We studied the effect of UTP on Schwannoma cell migration and wound closure and the intracellular signaling pathways involved. We found that UTP treatment induced Schwannoma cell migration through activation of P2Y2 receptors and through the increase of extracellular matrix metalloproteinase-2 (MMP-2) activation and expression. Knockdown P2Y2 receptor or MMP-2 expression greatly reduced wound closure and MMP-2 activation induced by UTP. MMP-2 activation evoked by injury or UTP was also mediated by phosphorylation of all 3 major mitogen-activated protein kinases (MAPKs): JNK, ERK1/2, and p38. Inhibition of these MAPK pathways decreased both MMP-2 activation and cell migration. Interestingly, MAPK phosphorylation evoked by UTP exhibited a biphasic pattern, with an early transient phosphorylation 5 min after treatment, and a late and sustained phosphorylation that appeared at 6 h and lasted up to 24 h. Inhibition of MMP-2 activity selectively blocked the late, but not the transient, phase of MAPK activation. These results suggest that MMP-2 activation and late MAPK phosphorylation are part of a positive feedback mechanism to maintain the migratory phenotype for wound healing. In conclusion, our findings show that treatment with UTP stimulates in vitro Schwannoma cell migration and wound repair through a MMP-2-dependent mechanism via P2Y2 receptors and MAPK pathway activation. PMID:24905332

Interleukin-6 expression under gravitational stress due to vibration and hypergravity in follicular thyroid cancer cells.

PubMed

Ma, Xiao; Wehland, Markus; Aleshcheva, Ganna; Hauslage, Jens; Waßer, Kai; Hemmersbach, Ruth; Infanger, Manfred; Bauer, Johann; Grimm, Daniela

2013-01-01

It is known that exposing cell lines in vitro to parabolic flights changes their gene expression and protein production patterns. Parabolic flights and spaceflight in general are accompanied by transient hypergravity and vibration, which may impact the cells and therefore, have to be considered too. To estimate the possible impact of transient hypergravity and vibration, we investigated the effects of these forces separately using dedicated ground-based facilities. We placed follicular thyroid ML-1 and CGTH W-1 cancer cells in a specific centrifuge (MuSIC Multi Sample Incubator Centrifuge; SAHC Short Arm Human Centrifuge) simulating the hypergravity phases that occur during one (P1) and 31 parabolas (P31) of parabolic flights, respectively. On the Vibraplex device, the same cell lines were treated with vibration waves corresponding to those that occur during a whole parabolic flight lasting for two hours. After the various treatments, cells were harvested and analyzed by quantitative real-time PCR, focusing on the genes involved in forming (ACTB, MYO9, TUBB, VIM, TLN1, and ITGB1) and modulating (EZR, RDX, and MSN) the cytoskeleton, as well as those encoding growth factors (EGF, CTGF, IL6, and IL8) or protein kinases (PRKAA1 and PRKCA). The analysis revealed alterations in several genes in both cell lines; however, fewer genes were affected in ML-1 than CGTH W-1 cells. Interestingly, IL6 was the only gene whose expression was changed in both cell lines by each treatment, while PKCA transcription remained unaffected in all experiments. We conclude that a PKCa-independent mechanism of IL6 gene activation is very sensitive to physical forces in thyroid cells cultured in vitro as monolayers.

Inactivation of IkappaBbeta by the tax protein of human T-cell leukemia virus type 1: a potential mechanism for constitutive induction of NF-kappaB.

PubMed

McKinsey, T A; Brockman, J A; Scherer, D C; Al-Murrani, S W; Green, P L; Ballard, D W

1996-05-01

In resting T lymphocytes, the transcription factor NF-kappaB is sequestered in the cytoplasm via interactions with members of the I kappa B family of inhibitors, including IkappaBalpha and IkappaBbeta. During normal T-cell activation, IkappaBalpha is rapidly phosphorylated, ubiquitinated, and degraded by the 26S proteasome, thus permitting the release of functional NF-kappaB. In contrast to its transient pattern of nuclear induction during an immune response, NF-kappaB is constitutively activated in cells expressing the Tax transforming protein of human T-cell leukemia virus type I (HTLV-1). Recent studies indicate that HTLV-1 Tax targets IkappaBalpha to the ubiquitin-proteasome pathway. However, it remains unclear how this viral protein induces a persistent rather than transient NF-kappaB response. In this report, we provide evidence that in addition to acting on IkappaBalpha, Tax stimulates the turnover Of IkappaBbeta via a related targeting mechanism. Like IkappaBalpha, Tax-mediated breakdown of IkappaBbeta in transfected T lymphocytes is blocked either by cell-permeable proteasome inhibitors or by mutation Of IkappaBbeta at two serine residues present within its N-terminal region. Despite the dual specificity of HTLV-1 Tax for IkappaBalpha and IkappaBbeta at the protein level, Tax selectively stimulates NF-kappaB-directed transcription of the IkappaBalpha gene. Consequently, IkappaBbeta protein expression is chronically downregulated in HTLV-1-infected T lymphocytes. These findings with IkappaBbeta provide a potential mechanism for the constitutive activation of NF-kappaB in Tax-expressing cells.

Interleukin-6 Expression under Gravitational Stress Due to Vibration and Hypergravity in Follicular Thyroid Cancer Cells

PubMed Central

Ma, Xiao; Wehland, Markus; Aleshcheva, Ganna; Hauslage, Jens; Waßer, Kai; Hemmersbach, Ruth; Infanger, Manfred; Bauer, Johann; Grimm, Daniela

2013-01-01

It is known that exposing cell lines in vitro to parabolic flights changes their gene expression and protein production patterns. Parabolic flights and spaceflight in general are accompanied by transient hypergravity and vibration, which may impact the cells and therefore, have to be considered too. To estimate the possible impact of transient hypergravity and vibration, we investigated the effects of these forces separately using dedicated ground-based facilities. We placed follicular thyroid ML-1 and CGTH W-1 cancer cells in a specific centrifuge (MuSIC Multi Sample Incubator Centrifuge; SAHC Short Arm Human Centrifuge) simulating the hypergravity phases that occur during one (P1) and 31 parabolas (P31) of parabolic flights, respectively. On the Vibraplex device, the same cell lines were treated with vibration waves corresponding to those that occur during a whole parabolic flight lasting for two hours. After the various treatments, cells were harvested and analyzed by quantitative real-time PCR, focusing on the genes involved in forming (ACTB, MYO9, TUBB, VIM, TLN1, and ITGB1) and modulating (EZR, RDX, and MSN) the cytoskeleton, as well as those encoding growth factors (EGF, CTGF, IL6, and IL8) or protein kinases (PRKAA1 and PRKCA). The analysis revealed alterations in several genes in both cell lines; however, fewer genes were affected in ML-1 than CGTH W-1 cells. Interestingly, IL6 was the only gene whose expression was changed in both cell lines by each treatment, while PKCA transcription remained unaffected in all experiments. We conclude that a PKCa-independent mechanism of IL6 gene activation is very sensitive to physical forces in thyroid cells cultured in vitro as monolayers. PMID:23844163

Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana.

PubMed

Karuppanan, Kalimuthu; Duhra-Gill, Sifti; Kailemia, Muchena J; Phu, My L; Lebrilla, Carlito B; Dandekar, Abhaya M; Rodriguez, Raymond L; Nandi, Somen; McDonald, Karen A

2017-01-04

Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 ( CMG2 ) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus ( CaMV ) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N -glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N -glycans consists of complex type structures in both protein samples. The most abundant (>50%) N -glycan structure was GlcNAc₂(Xy l )Man₃(Fuc)GlcNAc₂ in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N -glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting.

Identification of differentially regulated genes in human patent ductus arteriosus

PubMed Central

Parikh, Pratik; Bai, Haiqing; Swartz, Michael F; Alfieris, George M

2016-01-01

In order to identify differentially expressed genes that are specific to the ductus arteriosus, 18 candidate genes were evaluated in matched ductus arteriosus and aortic samples from infants with coarctation of the aorta. The cell specificity of the gene's promoters was assessed by performing transient transfection studies in primary cells derived from several patients. Segments of ductus arteriosus and aorta were isolated from infants requiring repair for coarctation of the aorta and used for mRNA quantitation and culturing of cells. Differences in expression were determined by quantitative PCR using the ΔΔCt method. Promoter regions of six of these genes were cloned into luciferase reporter plasmids for transient transfection studies in matched human ductus arteriosus and aorta cells. Transcription factor AP-2b and phospholipase A2 were significantly up-regulated in ductus arteriosus compared to aorta in whole tissues and cultured cells, respectively. In transient transfection experiments, Angiotensin II type 1 receptor and Prostaglandin E receptor 4 promoters consistently gave higher expression in matched ductus arteriosus versus aorta cells from multiple patients. Taken together, these results demonstrate that several genes are differentially expressed in ductus arteriosus and that their promoters may be used to drive ductus arteriosus-enriched transgene expression. PMID:27465141

Dual RNA regulatory control of a Staphylococcus aureus virulence factor.

PubMed

Chabelskaya, Svetlana; Bordeau, Valérie; Felden, Brice

2014-04-01

In pathogens, the accurate programming of virulence gene expression is essential for infection. It is achieved by sophisticated arrays of regulatory proteins and ribonucleic acids (sRNAs), but in many cases their contributions and connections are not yet known. Based on genetic, biochemical and structural evidence, we report that the expression pattern of a Staphylococcus aureus host immune evasion protein is enabled by the collaborative actions of RNAIII and small pathogenicity island RNA D (SprD). Their combined expression profiles during bacterial growth permit early and transient synthesis of Sbi to avoid host immune responses. Together, these two sRNAs use antisense mechanisms to monitor Sbi expression at the translational level. Deletion analysis combined with structural analysis of RNAIII in complex with its novel messenger RNA (mRNA) target indicate that three distant RNAIII domains interact with distinct sites of the sbi mRNA and that two locations are deep in the sbi coding region. Through distinct domains, RNAIII lowers production of two proteins required for avoiding innate host immunity, staphylococcal protein A and Sbi. Toeprints and in vivo mutational analysis reveal a novel regulatory module within RNAIII essential for attenuation of Sbi translation. The sophisticated translational control of mRNA by two differentially expressed sRNAs ensures supervision of host immune escape by a major pathogen.

The candidate sour taste receptor, PKD2L1, is expressed by type III taste cells in the mouse.

PubMed

Kataoka, Shinji; Yang, Ruibiao; Ishimaru, Yoshiro; Matsunami, Hiroaki; Sévigny, Jean; Kinnamon, John C; Finger, Thomas E

2008-03-01

The transient receptor potential channel, PKD2L1, is reported to be a candidate receptor for sour taste based on molecular biological and functional studies. Here, we investigated the expression pattern of PKD2L1-immunoreactivity (IR) in taste buds of the mouse. PKD2L1-IR is present in a few elongate cells in each taste bud as reported previously. The PKD2L1-expressing cells are different from those expressing PLCbeta2, a marker of Type II cells. Likewise PKD2L1-immunoreactive taste cells do not express ecto-ATPase which marks Type I cells. The PKD2L1-positive cells are immunoreactive for neural cell adhesion molecule, serotonin, PGP-9.5 (ubiquitin carboxy-terminal transferase), and chromogranin A, all of which are present in Type III taste cells. At the ultrastructural level, PKD2L1-immunoreactive cells form synapses onto afferent nerve fibers, another feature of Type III taste cells. These results are consistent with the idea that different taste cells in each taste bud perform distinct functions. We suggest that Type III cells are necessary for transduction and/or transmission of information about "sour", but have little or no role in transmission of taste information of other taste qualities.

The candidate sour taste receptor, PKD2L1, is expressed by type III taste cells in the mouse

PubMed Central

Kataoka, Shinji; Yang, Ruibiao; Ishimaru, Yoshiro; Matsunami, Hiroaki; Kinnamon, John C.; Finger, Thomas E.

2008-01-01

The transient receptor potential (TRP) channel, PKD2L1, is reported to be a candidate receptor for sour taste based on molecular biological and functional studies. Here, we investigated the expression pattern of PKD2L1-immunoreactivity (IR) in taste buds of the mouse. PKD2L1-IR is present in a few elongate cells in each taste bud as reported previously. The PKD2L1-expressing cells are different from those expressing PLCβ2, a marker of Type II cells. Likewise PKD2L1-immunoreactive taste cells do not express ecto-ATPase which marks Type I cells. The PKD2L1 positive cells are immunoreactive for NCAM, serotonin, PGP-9.5 (ubiquitin carboxy terminal transferase) and chromogranin A, all of which are present in Type III taste cells. At the ultrastructural level, PKD2L1-immunoreactive cells form synapses onto afferent nerve fibers, another feature of Type III taste cells. These results are consistent with the idea that different taste cells in each taste bud perform distinct functions. We suggest that Type III cells are necessary for transduction and/or transmission of information about “sour”, but have little or no role in transmission of taste information of other taste qualities. PMID:18156604

Pseudomonas aeruginosa elastase causes transient disruption of tight junctions and downregulation of PAR-2 in human nasal epithelial cells.

PubMed

Nomura, Kazuaki; Obata, Kazufumi; Keira, Takashi; Miyata, Ryo; Hirakawa, Satoshi; Takano, Ken-ichi; Kohno, Takayuki; Sawada, Norimasa; Himi, Tetsuo; Kojima, Takashi

2014-02-18

Pseudomonas aeruginosa causes chronic respiratory disease, and the elastase enzyme that it produces increases the permeability of airway epithelial cells owing to the disruption of tight junctions. P. aeruginosa is also implicated in prolonged chronic rhinosinusitis. However, the effects of P. aeruginosa elastase (PE) against the barrier formed by human nasal epithelial cells (HNECs) remain unknown. To investigate the mechanisms involved in the disruption of tight junctions by PE in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were used. The hTERT-HNECs were pretreated with inhibitors of various signal transduction pathways, PKC, MAPK, p38MAPK, PI3K, JNK, NF-κB, EGF receptor, proteasome, COX1 and COX2 before treatment with PE. Some cells were pretreated with siRNA and agonist of protease activated receptor-2 (PAR-2) before treatment with PE. Expression and structures of tight junctions were determined by Western blotting, real-time PCR, immunostaining and freeze-fracture. Transepithelial electrical resistance (TER) was examined as the epithelial barrier function. PE treatment transiently disrupted the epithelial barrier and downregulated the transmembrane proteins claudin-1 and -4, occludin, and tricellulin, but not the scaffold PDZ-expression proteins ZO-1 and -2 and adherens junction proteins E-cadherin and β-catenin. The transient downregulation of tight junction proteins was controlled via distinct signal transduction pathways such as the PKC, MAPK, PI3K, p38 MAPK, JNK, COX-1 and -2, and NF-κB pathways. Furthermore, treatment with PE transiently decreased PAR-2 expression, which also regulated the expression of the tight junction proteins. Treatment with a PAR-2 agonist prevented the downregulation of the tight junction proteins after PE treatment in HNECs. PE transiently disrupts tight junctions in HNECs and downregulates PAR-2. The transient disruption of tight junctions by PE might occur repeatedly during chronic rhinosinusitis.

Pseudomonas aeruginosa elastase causes transient disruption of tight junctions and downregulation of PAR-2 in human nasal epithelial cells

PubMed Central

2014-01-01

Background Pseudomonas aeruginosa causes chronic respiratory disease, and the elastase enzyme that it produces increases the permeability of airway epithelial cells owing to the disruption of tight junctions. P. aeruginosa is also implicated in prolonged chronic rhinosinusitis. However, the effects of P. aeruginosa elastase (PE) against the barrier formed by human nasal epithelial cells (HNECs) remain unknown. Methods To investigate the mechanisms involved in the disruption of tight junctions by PE in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were used. The hTERT-HNECs were pretreated with inhibitors of various signal transduction pathways, PKC, MAPK, p38MAPK, PI3K, JNK, NF-κB, EGF receptor, proteasome, COX1 and COX2 before treatment with PE. Some cells were pretreated with siRNA and agonist of protease activated receptor-2 (PAR-2) before treatment with PE. Expression and structures of tight junctions were determined by Western blotting, real-time PCR, immunostaining and freeze-fracture. Transepithelial electrical resistance (TER) was examined as the epithelial barrier function. Results PE treatment transiently disrupted the epithelial barrier and downregulated the transmembrane proteins claudin-1 and -4, occludin, and tricellulin, but not the scaffold PDZ-expression proteins ZO-1 and -2 and adherens junction proteins E-cadherin and β-catenin. The transient downregulation of tight junction proteins was controlled via distinct signal transduction pathways such as the PKC, MAPK, PI3K, p38 MAPK, JNK, COX-1 and -2, and NF-κB pathways. Furthermore, treatment with PE transiently decreased PAR-2 expression, which also regulated the expression of the tight junction proteins. Treatment with a PAR-2 agonist prevented the downregulation of the tight junction proteins after PE treatment in HNECs. Conclusions PE transiently disrupts tight junctions in HNECs and downregulates PAR-2. The transient disruption of tight junctions by PE might occur repeatedly during chronic rhinosinusitis. PMID:24548792

Unilateral or bilateral punctate hippocampal hyperintensities on DW-MRI: seizures, amnesia, or both?

PubMed

Bocos-Portillo, Jone; Escalza-Cortina, Inés; Gómez-Beldarrain, Marian; Rodriguez-Sainz, Aida; Garcia-Monco, Juan Carlos

2018-06-02

The presence of small hippocampal hyperintense lesions on diffusion-weighted (DW) MRI can respond to different etiologies and represents a challenge where clinical judgment is imperative, since therapeutic approach may be quite different.We here report three patients with similar neuroradiological findings, i.e., hyperintense punctate hippocampal lesions on diffusion-weighted MRI sequences, yet of different origin. The first one presented with isolated amnesia (transient global amnesia), the second one with amnesia and seizures, and the third one with seizures.Thus, hippocampal punctate lesions appear after transient global amnesia, but the same pattern may be present after seizures, either focal-onset or generalized seizures. This peculiar radiological MRI pattern could indicate a pathogenic link between transient global amnesia (TGA) and seizures which should be further studied.

Spaceflight induces both transient and heritable alterations in DNA methylation and gene expression in rice (Oryza sativa L.).

PubMed

Ou, Xiufang; Long, Likun; Zhang, Yunhong; Xue, Yiqun; Liu, Jingchun; Lin, Xiuyun; Liu, Bao

2009-03-09

Spaceflight represents a complex environmental condition in which several interacting factors such as cosmic radiation, microgravity and space magnetic fields are involved, which may provoke stress responses and jeopardize genome integrity. Given the inherent property of epigenetic modifications to respond to intrinsic as well as external perturbations, it is conceivable that epigenetic markers like DNA methylation may undergo alterations in response to spaceflight. We report here that extensive alteration in both DNA methylation and gene expression occurred in rice plants subjected to a spaceflight, as revealed by a set of characterized sequences including 6 transposable elements (TEs) and 11 cellular genes. We found that several features characterize the alterations: (1) All detected alterations are hypermethylation events; (2) whereas alteration in both CG and CNG methylation occurred in the TEs, only alteration in CNG methylation occurred in the cellular genes; (3) alteration in expression includes both up- and down-regulations, which did not show a general correlation with alteration in methylation; (4) altered methylation patterns in both TEs and cellular genes are heritable to progenies at variable frequencies; however, stochastic reversion to wild-type patterns and further de novo changes in progenies are also apparent; and (5) the altered expression states in both TEs and cellular genes are also heritable to selfed progenies but with markedly lower transmission frequencies than altered DNA methylation states. Furthermore, we found that a set of genes encoding for the various putative DNA methyltransferases, 5-methylcytosine DNA glycosylases, the SWI/SNF chromatin remodeller (DDM1) and siRNA-related proteins are extremely sensitive to perturbation by spaceflight, which might be an underlying cause for the altered methylation patterns in the space-flown plants. We discuss implications of spaceflight-induced epigenetic variations with regard to health safety issues of spaceship crews and potentiality of spaceflight as a means for mutagenesis in crop breeding.

Parthenolide accumulation and expression of genes related to parthenolide biosynthesis affected by exogenous application of methyl jasmonate and salicylic acid in Tanacetum parthenium.

PubMed

Majdi, Mohammad; Abdollahi, Mohammad Reza; Maroufi, Asad

2015-11-01

Up-regulation of germacrene A synthase and down-regulation of parthenolide hydroxylase genes play key role in parthenolide accumulation of feverfew plants treated with methyl jasmonate and salicylic acid. Parthenolide is an important sesquiterpene lactone due to its anti-migraine and anti-cancer properties. Parthenolide amount was quantified by high-performance liquid chromatography after foliar application of methyl jasmonate (100 µM) or salicylic acid (1.0 mM) on feverfew leaves in time course experiment (3-96 h). Results indicate that exogenous application of methyl jasmonate or salicylic acid activated parthenolide biosynthesis. Parthenolide content reached its highest amount at 24 h after methyl jasmonate or salicylic acid treatments, which were 3.1- and 1.96-fold higher than control plants, respectively. Parthenolide transiently increased due to methyl jasmonate or salicylic acid treatments until 24 h, but did not show significant difference compared with control plants at 48 and 96 h time points in both treatments. Also, the transcript levels of early pathway (upstream) genes of terpene biosynthesis including 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase and hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase and the biosynthetic genes of parthenolide including germacrene A synthase, germacrene A oxidase, costunolide synthase and parthenolide synthase were increased by methyl jasmonate and salicylic acid treatments, but with different intensity. The transcriptional levels of these genes were higher in methyl jasmonate-treated plants than salicylic acid-treated plants. Parthenolide content measurements along with expression pattern analysis of the aforementioned genes and parthenolide hydroxylase as side branch gene of parthenolide suggest that the expression patterns of early pathway genes were not directly consistent with parthenolide accumulation pattern; hence, parthenolide accumulation is probably further modulated by the expression of its biosynthetic genes, especially germacrene A synthase and also its side branch gene, parthenolide hydroxylase.

Analysis of transient hypermorphic activity of E(spl)D during R8 specification

PubMed Central

Majot, Adam T.

2017-01-01

Drosophila atonal (ato) is required for the specification of founding R8 photoreceptors during retinal development. ato is regulated via dual eye-specific enhancers; ato-3’ is subject to initial induction whereas 5’-ato facilitates Notch-mediated autoregulation. Notch is further utilized to induce bHLH repressors of the E(spl) locus to restrict Ato from its initial broad expression to individual cells. Although Notch operates in two, distinct phases, it has remained unclear how the two phases maintain independence from one another. The difference in these two phases has attributed to the hypothesized delayed expression of E(spl). However, immunofluorescence data indicate that E(spl) are expressed during early Ato patterning, suggesting a more sophisticated underlying mechanism. To probe this mechanism, we provide evidence that although E(spl) exert no influence on ato-3’, E(spl) repress 5’-ato and deletion of the E(spl) locus elicits precocious 5’-ato activity. Thus, E(spl) imposes a delay to the timing in which Ato initiates autoregulation. We next sought to understand this finding in the context of E(spl)D, which encodes a dysregulated variant of E(spl)M8 that perturbs R8 patterning, though, as previously reported, only in conjunction with the mutant receptor Nspl. We established a genetic interaction between E(spl)D and roughened eye (roe), a known modulator of Notch signaling in retinogenesis. This link further suggests a dosage-dependence between E(spl) and the proneural activators Ato and Sens, as indicated via interaction assays in which E(spl)D renders aberrant R8 patterning in conjunction with reduced proneural dosage. In total, the biphasicity of Notch signaling relies, to some degree, on the post-translational regulation of individual E(spl) members and, importantly, that post-translational regulation is likely necessary to modulate the level of E(spl) activity throughout the progression of Ato expression. PMID:29036187

Sildenafil prevents the up-regulation of transient receptor potential canonical channels in the development of cardiomyocyte hypertrophy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kiso, Hironori; Ohba, Takayoshi; Iino, Kenji

2013-07-05

Highlights: •Transient receptor potential canonical (TRPC1, 3 and 6) are up-regulated by ET-1. •Sildenafil inhibited hypertrophic responses (BNP, Ca entry, NFAT activation). •Sildenafil suppressed TRPC1, 3 and 6 expression. -- Abstract: Background: Transient receptor potential canonical (TRPCs) channels are up-regulated in the development of cardiac hypertrophy. Sildenafil inhibits TRPC6 activation and expression, leading to the prevention of cardiac hypertrophy. However, the effects of sildenafil on the expression of other TRPCs remain unknown. We hypothesized that in addition to its effects of TRPC6, sildenafil blocks the up-regulation of other TRPC channels to suppress cardiomyocyte hypertrophy. Methods and results: In cultured neonatalmore » rat cardiomyocytes, a 48 h treatment with 10 nM endothelin (ET)-1 induced hypertrophic responses characterized by nuclear factor of activated T cells activation and enhancement of brain natriuretic peptide expression and cell surface area. Co-treatment with sildenafil (1 μM, 48 h) inhibited these ET-1-induced hypertrophic responses. Although ET-1 enhanced the gene expression of TRPCs, sildenafil inhibited the enhanced gene expression of TRPC1, C3 and C6. Moreover, co-treatment with sildenafil abolished the augmentation of SOCE in the hypertrophied cardiomyocytes. Conclusions: These results suggest that sildenafil inhibits cardiomyocyte hypertrophy by suppressing the up-regulation of TRPC expression.« less

Electroporation transiently decreases GJB2 (connexin 26) expression in B16/BL6 melanoma cell line.

PubMed

Rangel, Marcelo Monte Mór; Chaible, Lucas Martins; Nagamine, Marcia Kazumi; Mennecier, Gregory; Cogliati, Bruno; de Oliveira, Krishna Duro; Fukumasu, Heidge; Sinhorini, Idércio Luiz; Mir, Lluis Maria; Dagli, Maria Lúcia Zaidan

2015-02-01

Connexins are proteins that form gap junctions. Perturbations in the cell membrane reportedly promote changes in the expression profile of connexins. Electroporation promotes destabilization by applying electrical pulses, and this procedure is used in electrochemotherapy and gene therapy, among others. This in vitro work aimed to study the interference of electroporation on the expression profile of GJB2 (Cx26 gene) and Connexin 26 in melanoma cell line B16/BL6. The techniques of immunocytochemistry, Western blot, and real-time PCR were used. After electroporation, cells showed a transient decrease in GJB2 mRNA. The immunostaining of Cx26 showed no noticeable change after electroporation at different time points. However, Western blot showed a significant reduction in Cx26 30 min after electroporation. Our results showed that electroporation interferes transiently in the expression of Connexin 26 in melanoma and are consistent with the idea that electroporation is a process of intense stress that promotes cell homeostatic imbalance and results in disruption of cell physiological processes such as transcription and translation.

Nuclei of Tsuga canadensis: Role of Flavanols in Chromatin Organization

PubMed Central

Feucht, Walter; Schmid, Markus; Treutter, Dieter

2011-01-01

Needle primordia of Tsuga canadensis (hemlock) arising from flank meristems of a shoot apex, form cell lineages consisting of four or eight cells. Within a recently established lineage there is striking uniformity in the pattern of nuclear flavanols. This fact points to an identical transcriptional expression of these flavanols during cell cycling. However two lineages, even if located close together within the same meristem, can be very different in the expression of both cell shape and nuclear flavanol pattern, indicating that epigenetic positional signals are operating in a collective specification of cell lineage development. There is a wide range of nuclear flavanol patterning from a mosaic-like distribution in an activated cell type to a homogenous appearance in silenced cell types. Single cells deriving from lineages are desynchronized because they underlie a signaling network at a higher tissue level which results in stronger epigenetic modifications of their nuclear flavanols. As an extreme case of epigenetic modulation, transient drought conditions caused a drastic reduction of nuclear flavanols. Upon treatment with sucrose or cytokinin, these nuclear flavanols could be fully restored. Analytical determination of the flavanols revealed 3.4 mg/g DW for newly sprouting needles and 19.6 mg/g DW for anthers during meiosis. The roughly 6-fold difference in flavanols is apparently a reflection of the highly diverging organogenetic processes. Collectively, the studies provide strong evidence for combinatorial interplay between cell fate and nuclear flavanols. PMID:22072922

Transient receptor potential ion channels control thermoregulatory behaviour in reptiles.

PubMed

Seebacher, Frank; Murray, Shauna A

2007-03-14

Biological functions are governed by thermodynamics, and animals regulate their body temperature to optimise cellular performance and to avoid harmful extremes. The capacity to sense environmental and internal temperatures is a prerequisite for the evolution of thermoregulation. However, the mechanisms that enable ectothermic vertebrates to sense heat remain unknown. The recently discovered thermal characteristics of transient receptor potential ion channels (TRP) render these proteins suitable to act as temperature sensors. Here we test the hypothesis that TRPs are present in reptiles and function to control thermoregulatory behaviour. We show that the hot-sensing TRPV1 is expressed in a crocodile (Crocodylus porosus), an agamid (Amphibolurus muricatus) and a scincid (Pseudemoia entrecasteauxii) lizard, as well as in the quail and zebrafinch (Coturnix chinensis and Poephila guttata). The TRPV1 genes from all reptiles form a unique clade that is delineated from the mammalian and the ancestral Xenopus sequences by an insertion of two amino acids. TRPV1 and the cool-sensing TRPM8 are expressed in liver, muscle (transversospinalis complex), and heart tissues of the crocodile, and have the potential to act as internal thermometer and as external temperatures sensors. Inhibition of TRPV1 and TRPM8 in C. porosus abolishes the typically reptilian shuttling behaviour between cooling and heating environments, and leads to significantly altered body temperature patterns. Our results provide the proximate mechanism of thermal selection in terrestrial ectotherms, which heralds a fundamental change in interpretation, because TRPs provide the mechanism for a tissue-specific input into the animals' thermoregulatory response.

Tightly Regulated Expression of Autographa californica Multicapsid Nucleopolyhedrovirus Immediate Early Genes Emerges from Their Interactions and Possible Collective Behaviors

PubMed Central

Taka, Hitomi; Asano, Shin-ichiro; Matsuura, Yoshiharu; Bando, Hisanori

2015-01-01

To infect their hosts, DNA viruses must successfully initiate the expression of viral genes that control subsequent viral gene expression and manipulate the host environment. Viral genes that are immediately expressed upon infection play critical roles in the early infection process. In this study, we investigated the expression and regulation of five canonical regulatory immediate-early (IE) genes of Autographa californica multicapsid nucleopolyhedrovirus: ie0, ie1, ie2, me53, and pe38. A systematic transient gene-expression analysis revealed that these IE genes are generally transactivators, suggesting the existence of a highly interactive regulatory network. A genetic analysis using gene knockout viruses demonstrated that the expression of these IE genes was tolerant to the single deletions of activator IE genes in the early stage of infection. A network graph analysis on the regulatory relationships observed in the transient expression analysis suggested that the robustness of IE gene expression is due to the organization of the IE gene regulatory network and how each IE gene is activated. However, some regulatory relationships detected by the genetic analysis were contradictory to those observed in the transient expression analysis, especially for IE0-mediated regulation. Statistical modeling, combined with genetic analysis using knockout alleles for ie0 and ie1, showed that the repressor function of ie0 was due to the interaction between ie0 and ie1, not ie0 itself. Taken together, these systematic approaches provided insight into the topology and nature of the IE gene regulatory network. PMID:25816136

Heterogeneity of transverse-axial tubule system in mouse atria: Remodeling in atrial-specific Na+-Ca2+ exchanger knockout mice.

PubMed

Yue, Xin; Zhang, Rui; Kim, Brian; Ma, Aiqun; Philipson, Kenneth D; Goldhaber, Joshua I

2017-07-01

Transverse-axial tubules (TATs) are commonly assumed to be sparse or absent in atrial myocytes from small animals. Atrial myocytes from rats, cats and rabbits lack TATs, which results in a characteristic "V"-shaped Ca release pattern in confocal line-scan recordings due to the delayed rise of Ca in the center of the cell. To examine TAT expression in isolated mouse atrial myocytes, we loaded them with the membrane dye Di-4-ANEPPS to label TATs. We found that >80% of atrial myocytes had identifiable TATs. Atria from male mice had a higher TAT density than female mice, and TAT density correlated with cell width. Using the fluorescent Ca indicator Fluo-4-AM and confocal imaging, we found that wild type (WT) mouse atrial myocytes generate near-synchronous Ca transients, in contrast to the "V"-shaped pattern typically reported in other small animals such as rat. In atrial-specific Na-Ca exchanger (NCX) knockout (KO) mice, which develop sinus node dysfunction and atrial hypertrophy with dilation, we found a substantial loss of atrial TATs in isolated atrial myocytes. There was a greater loss of transverse tubules compared to axial tubules, resulting in a dominance of axial tubules. Consistent with the overall loss of TATs, NCX KO atrial myocytes displayed a "V"-shaped Ca transient with slower and reduced central (CT) Ca release and uptake in comparison to subsarcolemmal (SS) Ca release. We compared chemically detubulated (DT) WT cells to KO, and found similar slowing of CT Ca release and uptake. However, SS Ca transients in the WT DT cells had faster uptake kinetics than KO cells, consistent with the presence of NCX and normal sarcolemmal Ca efflux in the WT DT cells. We conclude that the remodeling of NCX KO atrial myocytes is accompanied by a loss of TATs leading to abnormal Ca release and uptake that could impact atrial contractility and rhythm. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transient left septal and anterior fascicular block associated with type 1 electrocardiographic Brugada pattern.

PubMed

Pérez-Riera, Andrés Ricardo; Barbosa-Barros, Raimundo; Penachini da Costa de Rezende Barbosa, Marianne; Daminello-Raimundo, Rodrigo; de Abreu, Luiz Carlos

The left septal fascicular block (LSFB) or blockage of the middle fibers of the left bundle branch is probably caused mainly by - in the developed world - the proximal obstruction of the left anterior descending artery (LAD) before its first anterior septal perforator branch (S 1 ). The association of transient LSFB and left anterior fascicular block (LAFB) - left bifascicular block - and the electrocardiographic type 1 Brugada pattern (BrP) has not been described in the literature yet. Copyright © 2017 Elsevier Inc. All rights reserved.

Ischemic preconditioning protects neurons from damage and maintains the immunoreactivity of kynurenic acid in the gerbil hippocampal CA1 region following transient cerebral ischemia

PubMed Central

LEE, JAE-CHUL; TAE, HYUN-JIN; CHO, GEUM-SIL; KIM, IN HYE; AHN, JI HYEON; PARK, JOON HA; CHEN, BAI HUI; CHO, JEONG-HWI; SHIN, BICH NA; CHO, JUN HWI; BAE, EUN JOO; PARK, JINSEU; KIM, YOUNG-MYEONG; CHOI, SOO YOUNG; WON, MOO-HO

2015-01-01

Pyramidal neurons in region I of hippocampus proper (CA1) are particularly vulnerable to excitotoxic processes following transient forebrain ischemia. Kynurenic acid (KYNA) is a small molecule derived from tryptophan when this amino acid is metabolized through the kynurenine pathway. In the present study, we examined the effects of ischemic preconditioning (IPC) on the immunoreactivity and protein levels of KYNA following 5 min of transient forebrain ischemia in gerbils. The animals were randomly assigned to 4 groups (sham-operated group, ischemia-operated group, IPC + sham-operated group and IPC + ischemia-operated group). IPC was induced by subjecting the gerbils to 2 min of ischemia followed by 1 day of recovery. In the ischemia-operated group, we observed a significant loss of pyramidal neurons in the CA1 stratum pyramidale (SP) at 5 days post-ischemia; however, in the IPC + ischemia-operated group, the pyramidal neurons were well protected. KYNA immunoreactivity in the SP of the ischemia-operated group was significantly altered following ischemia-reperfusion and was very low 5 days following ischemia-reperfusion. In the IPC + ischemia-operated group, however, KYNA immunoreactivity was constitutively detected in the SP of the CA1 region after the ischemic insult. We also found that the alteration pattern of the KYNA protein level in the CA1 region following ischemia was generally similar to the immunohistochemical changes observed. In brief, our findings demonstrated that IPC maintained and even increased KYNA immunoreactivity in the SP of the CA1 region following ischemia-reperfusion. The data from the present study thus indicate that the enhancement of KYNA expression by IPC may be necessary for neuronal survival following transient ischemic injury. PMID:25872573

Metabolic studies of transient tyrosinemia in premature infants

NASA Technical Reports Server (NTRS)

Fernbach, S. A.; Summons, R. E.; Pereira, W. E.; Duffield, A. M.

1975-01-01

The recently developed technique of gas chromatography-mass spectrometry supported by computer has considerably improved the analysis of physiologic fluids. This study attempted to demonstrate the value of this system in the investigation of metabolite patterns in urine in two metabolic problems of prematurity, transient tyrosinemia and late metabolic acidosis. Serial 24-hr urine specimens were analyzed in 9 infants. Transient tyrosinemia, characterized by 5- 10-fold increases over basal excretion of tyrosine, p-hydroxyphenyllactate, and p-hydroxyphenylpyruvate in urine, was noted in five of the infants. Late metabolic acidosis was seen in four infants, but bore no relation to transient tyrosinemia.

Cerebral Cortex Hyperthyroidism of Newborn Mct8-Deficient Mice Transiently Suppressed by Lat2 Inactivation

PubMed Central

Núñez, Bárbara; Martínez de Mena, Raquel; Obregon, Maria Jesus; Font-Llitjós, Mariona; Nunes, Virginia; Palacín, Manuel; Dumitrescu, Alexandra M.; Morte, Beatriz; Bernal, Juan

2014-01-01

Thyroid hormone entry into cells is facilitated by transmembrane transporters. Mutations of the specific thyroid hormone transporter, MCT8 (Monocarboxylate Transporter 8, SLC16A2) cause an X-linked syndrome of profound neurological impairment and altered thyroid function known as the Allan-Herndon-Dudley syndrome. MCT8 deficiency presumably results in failure of thyroid hormone to reach the neural target cells in adequate amounts to sustain normal brain development. However during the perinatal period the absence of Mct8 in mice induces a state of cerebral cortex hyperthyroidism, indicating increased brain access and/or retention of thyroid hormone. The contribution of other transporters to thyroid hormone metabolism and action, especially in the context of MCT8 deficiency is not clear. We have analyzed the role of the heterodimeric aminoacid transporter Lat2 (Slc7a8), in the presence or absence of Mct8, on thyroid hormone concentrations and on expression of thyroid hormone-dependent cerebral cortex genes. To this end we generated Lat2-/-, and Mct8-/yLat2 -/- mice, to compare with wild type and Mct8-/y mice during postnatal development. As described previously the single Mct8 KO neonates had a transient increase of 3,5,3′-triiodothyronine concentration and expression of thyroid hormone target genes in the cerebral cortex. Strikingly the absence of Lat2 in the double Mct8Lat2 KO prevented the effect of Mct8 inactivation in newborns. The Lat2 effect was not observed from postnatal day 5 onwards. On postnatal day 21 the Mct8 KO displayed the typical pattern of thyroid hormone concentrations in plasma, decreased cortex 3,5,3′-triiodothyronine concentration and Hr expression, and concomitant Lat2 inactivation produced little to no modifications. As Lat2 is expressed in neurons and in the choroid plexus, the results support a role for Lat2 in the supply of thyroid hormone to the cerebral cortex during early postnatal development. PMID:24819605

Cerebral cortex hyperthyroidism of newborn mct8-deficient mice transiently suppressed by lat2 inactivation.

PubMed

Núñez, Bárbara; Martínez de Mena, Raquel; Obregon, Maria Jesus; Font-Llitjós, Mariona; Nunes, Virginia; Palacín, Manuel; Dumitrescu, Alexandra M; Morte, Beatriz; Bernal, Juan

2014-01-01

Thyroid hormone entry into cells is facilitated by transmembrane transporters. Mutations of the specific thyroid hormone transporter, MCT8 (Monocarboxylate Transporter 8, SLC16A2) cause an X-linked syndrome of profound neurological impairment and altered thyroid function known as the Allan-Herndon-Dudley syndrome. MCT8 deficiency presumably results in failure of thyroid hormone to reach the neural target cells in adequate amounts to sustain normal brain development. However during the perinatal period the absence of Mct8 in mice induces a state of cerebral cortex hyperthyroidism, indicating increased brain access and/or retention of thyroid hormone. The contribution of other transporters to thyroid hormone metabolism and action, especially in the context of MCT8 deficiency is not clear. We have analyzed the role of the heterodimeric aminoacid transporter Lat2 (Slc7a8), in the presence or absence of Mct8, on thyroid hormone concentrations and on expression of thyroid hormone-dependent cerebral cortex genes. To this end we generated Lat2-/-, and Mct8-/yLat2-/- mice, to compare with wild type and Mct8-/y mice during postnatal development. As described previously the single Mct8 KO neonates had a transient increase of 3,5,3'-triiodothyronine concentration and expression of thyroid hormone target genes in the cerebral cortex. Strikingly the absence of Lat2 in the double Mct8Lat2 KO prevented the effect of Mct8 inactivation in newborns. The Lat2 effect was not observed from postnatal day 5 onwards. On postnatal day 21 the Mct8 KO displayed the typical pattern of thyroid hormone concentrations in plasma, decreased cortex 3,5,3'-triiodothyronine concentration and Hr expression, and concomitant Lat2 inactivation produced little to no modifications. As Lat2 is expressed in neurons and in the choroid plexus, the results support a role for Lat2 in the supply of thyroid hormone to the cerebral cortex during early postnatal development.

Hilar Mossy Cell Degeneration Causes Transient Dentate Granule Cell Hyperexcitability and Impaired Pattern Separation

PubMed Central

Jinde, Seiichiro; Zsiros, Veronika; Jiang, Zhihong; Nakao, Kazuhito; Pickel, James; Kohno, Kenji; Belforte, Juan E.; Nakazawa, Kazu

2012-01-01

Summary Although excitatory mossy cells of the hippocampal hilar region are known to project both to dentate granule cells and to interneurons, it is as yet unclear whether mossy cell activity’s net effect on granule cells is excitatory or inhibitory. To explore their influence on dentate excitability and hippocampal function, we generated a conditional transgenic mouse line, using the Cre/loxP system, in which diphtheria toxin receptor was selectively expressed in mossy cells. One week after injecting toxin into this line, mossy cells throughout the longitudinal axis were degenerated extensively, theta wave power of dentate local field potentials increased during exploration, and deficits occurred in contextual discrimination. By contrast, we detected no epileptiform activity, spontaneous behavioral seizures, or mossy-fiber sprouting 5–6 weeks after mossy cell degeneration. These results indicate that the net effect of mossy cell excitation is to inhibit granule cell activity and enable dentate pattern separation. PMID:23259953

Role of TRP ion channels in cancer and tumorigenesis.

PubMed

Shapovalov, George; Ritaine, Abigael; Skryma, Roman; Prevarskaya, Natalia

2016-05-01

Transient receptor potential (TRP) channels are recently identified proteins that form a versatile family of ion channels, the majority of which are calcium permeable and exhibit complex regulatory patterns with sensitivity to multiple environmental factors. While this sensitivity has captured early attention, leading to recognition of TRP channels as environmental and chemical sensors, many later studies concentrated on the regulation of intracellular calcium by TRP channels. Due to mutations, dysregulation of ion channel gating or expression levels, normal spatiotemporal patterns of local Ca(2+) distribution become distorted. This causes deregulation of downstream effectors sensitive to changes in Ca(2+) homeostasis that, in turn, promotes pathophysiological cancer hallmarks, such as enhanced survival, proliferation and invasion. These observations give rise to the appreciation of the important contributions that TRP channels make to many cellular processes controlling cell fate and positioning these channels as important players in cancer regulation. This review discusses the accumulated scientific knowledge focused on TRP channel involvement in regulation of cell fate in various transformed tissues.

Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

PubMed Central

Ngô, V; Gourdji, D; Laverrière, J N

1996-01-01

The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo. PMID:8668139

Modeling coexistence of oscillation and Delta/Notch-mediated lateral inhibition in pancreas development and neurogenesis.

PubMed

Tiedemann, Hendrik B; Schneltzer, Elida; Beckers, Johannes; Przemeck, Gerhard K H; Hrabě de Angelis, Martin

2017-10-07

During pancreas development, Neurog3 positive endocrine progenitors are specified by Delta/Notch (D/N) mediated lateral inhibition in the growing ducts. During neurogenesis, genes that determine the transition from the proneural state to neuronal or glial lineages are oscillating before their expression is sustained. Although the basic gene regulatory network is very similar, cycling gene expression in pancreatic development was not investigated yet, and previous simulations of lateral inhibition in pancreas development excluded by design the possibility of oscillations. To explore this possibility, we developed a dynamic model of a growing duct that results in an oscillatory phase before the determination of endocrine progenitors by lateral inhibition. The basic network (D/N + Hes1 + Neurog3) shows scattered, stable Neurog3 expression after displaying transient expression. Furthermore, we included the Hes1 negative feedback as previously discussed in neurogenesis and show the consequences for Neurog3 expression in pancreatic duct development. Interestingly, a weakened HES1 action on the Hes1 promoter allows the coexistence of stable patterning and oscillations. In conclusion, cycling gene expression and lateral inhibition are not mutually exclusive. In this way, we argue for a unified mode of D/N mediated lateral inhibition in neurogenic and pancreatic progenitor specification. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

How gastrin-releasing peptide receptor (GRPR) and αvβ3 integrin expression reflect reorganization features of tumors after hyperthermia treatments.

PubMed

Hallasch, Sandra; Frick, Sindy; Jung, Maximilian; Hilger, Ingrid

2017-07-31

The outcome of tumor treatment via hyperthermia in the clinic has been reported to be heterogeneous. Here, we assessed how the presence of gastrin-releasing peptide receptor (GRPR) and α v β 3 integrin together with the morphology of the vascularization reflects the growth behavior of tumors after hyperthermia treatment. MDA-MB-231 tumor bearing mice were treated either with high (46 °C) or low dose (42 °C) water hyperthermia for 60 min. Changes of GRPR and α v β 3 integrin expression were assessed via multiplexed optical imaging. Vascularization was reconstructed and quantified by µCT imaging after contrast agent injection. We found that high dose hyperthermia is capable of increasing the expression of GRPR, α v β 3 integrin, CD31, and Ki67 in tumors. Also the morphology of tumor vasculature changed (increased relative blood volume and small-diameter vessel density, decreased expression of α-SMA). Low dose hyperthermia induced comparatively moderate effects on the investigated protein expression pattern and vascular remodeling. We conclude that under defined circumstances, specific temperature doses affect the reorganization of tumor regrowth, which is triggered by residual "dormant" cells even though tumor volumes are transiently decreasing. Further on, GRPR, α v β 3 integrin expression are versatile tools to surveil potential tumor regrow during therapy, beyond the conventional determination of tumor volumes.

Correlation between Urothelial Differentiation and Sensory Proteins P2X3, P2X5, TRPV1, and TRPV4 in Normal Urothelium and Papillary Carcinoma of Human Bladder

PubMed Central

Sterle, Igor; Zupančič, Daša; Romih, Rok

2014-01-01

Terminal differentiation of urothelium is a prerequisite for blood-urine barrier formation and enables normal sensory function of the urinary bladder. In this study, urothelial differentiation of normal human urothelium and of low and high grade papillary urothelial carcinomas was correlated with the expression and localization of purinergic receptors (P2X3, and P2X5) and transient receptor potential vanilloid channels (TRPV1, and TRPV4). Western blotting and immunofluorescence of uroplakins together with scanning electron microscopy of urothelial apical surface demonstrated terminal differentiation of normal urothelium, partial differentiation of low grade carcinoma, and poor differentiation of high grade carcinoma. P2X3 was expressed in normal urothelium as well as in low grade carcinoma and in both cases immunolabeling was stronger in the superficial cells. P2X3 expression decreased in high grade carcinoma. P2X5 expression was detected in normal urothelium and in high grade carcinoma, while in low grade carcinoma its expression was diminished. The expression of TRPV1 decreased in low grade and even more in high grade carcinoma when compared with normal urothelium, while TRPV4 expression was unchanged in all samples. Our results suggest that sensory proteins P2X3 and TRPV1 are in correlation with urothelial differentiation, while P2X5 and TRPV4 have unique expression patterns. PMID:24868547

Correlation between urothelial differentiation and sensory proteins P2X3, P2X5, TRPV1, and TRPV4 in normal urothelium and papillary carcinoma of human bladder.

PubMed

Sterle, Igor; Zupančič, Daša; Romih, Rok

2014-01-01

Terminal differentiation of urothelium is a prerequisite for blood-urine barrier formation and enables normal sensory function of the urinary bladder. In this study, urothelial differentiation of normal human urothelium and of low and high grade papillary urothelial carcinomas was correlated with the expression and localization of purinergic receptors (P2X3, and P2X5) and transient receptor potential vanilloid channels (TRPV1, and TRPV4). Western blotting and immunofluorescence of uroplakins together with scanning electron microscopy of urothelial apical surface demonstrated terminal differentiation of normal urothelium, partial differentiation of low grade carcinoma, and poor differentiation of high grade carcinoma. P2X3 was expressed in normal urothelium as well as in low grade carcinoma and in both cases immunolabeling was stronger in the superficial cells. P2X3 expression decreased in high grade carcinoma. P2X5 expression was detected in normal urothelium and in high grade carcinoma, while in low grade carcinoma its expression was diminished. The expression of TRPV1 decreased in low grade and even more in high grade carcinoma when compared with normal urothelium, while TRPV4 expression was unchanged in all samples. Our results suggest that sensory proteins P2X3 and TRPV1 are in correlation with urothelial differentiation, while P2X5 and TRPV4 have unique expression patterns.

Safety, immunogenicity, and efficacy of the ML29 reassortant vaccine for Lassa fever in small non-human primates✩

PubMed Central

Lukashevich, Igor S.; Carrion, Ricardo; Salvato, Maria S.; Mansfield, Keith; Brasky, Kathleen; Zapata, Juan; Cairo, Cristiana; Goicochea, Marco; Hoosien, Gia E.; Ticer, Anysha; Bryant, Joseph; Davis, Harry; Hammamieh, Rasha; Mayda, Maria; Jett, Marti; Patterson, Jean

2008-01-01

A single injection of ML29 reassortant vaccine for Lassa fever induces low, transient viremia, and low or moderate levels of ML29 replication in tissues of common marmosets depending on the dose of the vaccination. The vaccination elicits specific immune responses and completely protects marmosets against fatal disease by induction of sterilizing cell-mediated immunity. DNA array analysis of human peripheral blood mononuclear cells from healthy donors exposed to ML29 revealed that gene expression patterns in ML29-exposed PBMC and control, media-exposed PBMC, clustered together confirming safety profile of the ML29 in non-human primates. The ML29 reassortant is a promising vaccine candidate for Lassa fever. PMID:18692539

Transient Co-Expression of Post-Transcriptional Gene Silencing Suppressors for Increased in Planta Expression of a Recombinant Anthrax Receptor Fusion Protein

PubMed Central

Arzola, Lucas; Chen, Junxing; Rattanaporn, Kittipong; Maclean, James M.; McDonald, Karen A.

2011-01-01

Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin), CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA) domain of human capillary morphogenesis 2 (CMG2), an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG). We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc with nine different viral suppressors of post-transcriptional gene silencing (PTGS): p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI), with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI. PMID:21954339

One nuclear calcium transient induced by a single burst of action potentials represents the minimum signal strength in activity-dependent transcription in hippocampal neurons.

PubMed

Yu, Yan; Oberlaender, Kristin; Bengtson, C Peter; Bading, Hilmar

2017-07-01

Neurons undergo dramatic changes in their gene expression profiles in response to synaptic stimulation. The coupling of neuronal excitation to gene transcription is well studied and is mediated by signaling pathways activated by cytoplasmic and nuclear calcium transients. Despite this, the minimum synaptic activity required to induce gene expression remains unknown. To address this, we used cultured hippocampal neurons and cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH) that allows detection of nascent transcripts in the cell nucleus. We found that a single burst of action potentials, consisting of 24.4±5.1 action potentials during a 6.7±1.9s depolarization of 19.5±2.0mV causing a 9.3±0.9s somatic calcium transient, is sufficient to activate transcription of the immediate early gene arc (also known as Arg3.1). The total arc mRNA yield produced after a single burst-induced nuclear calcium transient was very small and, compared to unstimulated control neurons, did not lead to a significant increase in arc mRNA levels measured using quantitative reverse transcriptase PCR (qRT-PCR) of cell lysates. Significantly increased arc mRNA levels became detectable in hippocampal neurons that had undergone 5-8 consecutive burst-induced nuclear calcium transients at 0.05-0.15Hz. These results indicate that a single burst-induced nuclear calcium transient can activate gene expression and that transcription is rapidly shut off after synaptic stimulation has ceased. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of nutrient deprivation and differentiation on the expression of growth-arrest genes (gas and gadd) in F9 embryonal carcinoma cells.

PubMed Central

Fleming, J V; Hay, S M; Harries, D N; Rees, W D

1998-01-01

The growth-arrest genes (gas and gadd) are widely expressed during mammalian embryogenesis and may be useful as markers of nutritional stress in the embryo. F9 embryonal carcinoma cells have been used to characterize the effect of serum or amino acid deficiency on growth-arrest gene expression in a differentiating embryonic cell. The differentiation markers, homeobox B2 (HoxB2), collagen type IV and laminin B2, were not induced by growth arrest. Treatment with all-trans retinoic acid (RA) produced a dose-dependent increase in alkaline phosphatase activity, which was unchanged in lysine-deficient medium and reduced in low-serum medium. Low-serum medium also reduced HoxB2 expression. There was a transient 2-6-fold increase in mRNAs for C/EBP-beta, gadd153/CHOP-10 and gas5 genes 24 h after transfer to amino-acid-deficient media. The mRNAs for the gas2 and gas6 genes began to rise slowly by 5-10-fold after a delay of approx. 24 h. The transient increases did not occur in low-serum medium where there was a much smaller and slower increase. Differentiation caused 1-2-fold increases in gas2, gas3 and gas6 mRNA levels. The transient overexpression of gas5, gadd153/CHOP-10 and CCAAT-enhancer-binding protein-beta, and the later expression of gas6 mRNAs in response to amino acid deficiency, were not affected by differentiation. RA treatment increased the expression of gas3 and caused gas2 to be transiently overexpressed in amino-acid-deficient medium. Differentiation in serum-deficient medium did not significantly alter the levels of the growth-arrest gene mRNAs. These results show that in F9 cells the growth-arrest genes are expressed sequentially as a result of nutrient stress. PMID:9461558

Effects of nutrient deprivation and differentiation on the expression of growth-arrest genes (gas and gadd) in F9 embryonal carcinoma cells.

PubMed

Fleming, J V; Hay, S M; Harries, D N; Rees, W D

1998-02-15

The growth-arrest genes (gas and gadd) are widely expressed during mammalian embryogenesis and may be useful as markers of nutritional stress in the embryo. F9 embryonal carcinoma cells have been used to characterize the effect of serum or amino acid deficiency on growth-arrest gene expression in a differentiating embryonic cell. The differentiation markers, homeobox B2 (HoxB2), collagen type IV and laminin B2, were not induced by growth arrest. Treatment with all-trans retinoic acid (RA) produced a dose-dependent increase in alkaline phosphatase activity, which was unchanged in lysine-deficient medium and reduced in low-serum medium. Low-serum medium also reduced HoxB2 expression. There was a transient 2-6-fold increase in mRNAs for C/EBP-beta, gadd153/CHOP-10 and gas5 genes 24 h after transfer to amino-acid-deficient media. The mRNAs for the gas2 and gas6 genes began to rise slowly by 5-10-fold after a delay of approx. 24 h. The transient increases did not occur in low-serum medium where there was a much smaller and slower increase. Differentiation caused 1-2-fold increases in gas2, gas3 and gas6 mRNA levels. The transient overexpression of gas5, gadd153/CHOP-10 and CCAAT-enhancer-binding protein-beta, and the later expression of gas6 mRNAs in response to amino acid deficiency, were not affected by differentiation. RA treatment increased the expression of gas3 and caused gas2 to be transiently overexpressed in amino-acid-deficient medium. Differentiation in serum-deficient medium did not significantly alter the levels of the growth-arrest gene mRNAs. These results show that in F9 cells the growth-arrest genes are expressed sequentially as a result of nutrient stress.

Onset of Tlx-3 expression in the chick cerebellar cortex correlates with the morphological development of fissures and delineates a posterior transverse boundary.

PubMed

Logan, Cairine; Millar, Cassie; Bharadia, Vinay; Rouleau, Katherine

2002-06-24

Recent studies have shown that the mammalian cerebellar cortex can be subdivided into a reproducible array of zones and stripes. In particular, discontinuous patterns of gene expression together with mutational analysis suggest that there are at least four distinct transverse zones along the rostrocaudal axis in mouse: the anterior zone (lobules I-V), the central zone (lobules VI and VII), the posterior zone (lobules VIII and IX), and the nodular zone (lobule X). Here we show that the divergent homeobox-containing transcription factor, Tlx- 3 (also known as Hox11L2 or Rnx) is transiently expressed in external granule cells in a distinct transverse domain of the developing chick cerebellar cortex. Expression is first detected at Hamburger and Hamilton (HH) stage 35. Interestingly, Tlx-3 mRNA expression is initially confined to, and coincident with, the morphological development of fissures. Slightly later, at HH stage 38, expression extends throughout the developing external granular layer (EGL) of lobules I-IXab. Notably, no Tlx-3 expression was detected in lobules IXc and X at any developmental time point examined. Expression is noticeably stronger in nonproliferating cells located in the deep layer of the EGL. Tlx-3 expression is downregulated as granule cells migrate inward to form the internal granule layer and is undetectable shortly after birth. These results suggest that Tlx-3 is expressed as granule cells become postmitotic and suggest that Tlx-3 may play a role in the differentiation of distinct neuronal populations in the cerebellum. Copyright 2002 Wiley-Liss, Inc.

Assessment of Different Turbulence Models for the Motion of Non-metallic Inclusion in Induction Crucible Furnace

NASA Astrophysics Data System (ADS)

Barati, H.; Wu, M.; Kharicha, A.; Ludwig, A.

2016-07-01

Turbulent fluid flow due to the electromagnetic forces in induction crucible furnace (ICF) is modeled using k-ɛ, k-ω SST and Large Eddy Simulation (LES) turbulence models. Fluid flow patterns calculated by different turbulence models and their effects on the motion of non-metallic inclusions (NMI) in the bulk melt have been investigated. Results show that the conventional k-ɛ model cannot solve the transient flow in ICF properly. With k-ω model transient flow and oscillation behavior of the flow pattern can be solved, and the motion of NMI can be tracked fairly well. LES model delivers the best modeling result on both details of the transient flow pattern and motion trajectories of NMI without the limitation of NMI size. The drawback of LES model is the long calculation time. Therefore, for general purpose to estimate the dynamic behavior of NMI in ICF both k-ω SST and LES are recommended. For the precise calculation of the motion of NMI smaller than 10 μm only LES model is appropriate.

Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana.

PubMed

Huddy, Suzanne M; Hitzeroth, Inga I; Meyers, Ann E; Weber, Brandon; Rybicki, Edward P

2018-01-01

Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish ( Armoracia rusticana ); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N. benthamiana was evaluated using an Agrobacterium tumefaciens -mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in N. benthamiana in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme.

Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana

PubMed Central

Huddy, Suzanne M.; Hitzeroth, Inga I.; Weber, Brandon; Rybicki, Edward P.

2018-01-01

Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish (Armoracia rusticana); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N. benthamiana was evaluated using an Agrobacterium tumefaciens-mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in N. benthamiana in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme. PMID:29301255

Transient ectopic expression of the histone demethylase JMJD3 accelerates the differentiation of human pluripotent stem cells

PubMed Central

Wakabayashi, Shunichi; Soma, Atsumi; Sato, Saeko; Nakatake, Yuhki; Oda, Mayumi; Murakami, Miyako; Sakota, Miki; Chikazawa-Nohtomi, Nana

2016-01-01

Harnessing epigenetic regulation is crucial for the efficient and proper differentiation of pluripotent stem cells (PSCs) into desired cell types. Histone H3 lysine 27 trimethylation (H3K27me3) functions as a barrier against cell differentiation through the suppression of developmental gene expression in PSCs. Here, we have generated human PSC (hPSC) lines in which genome-wide reduction of H3K27me3 can be induced by ectopic expression of the catalytic domain of the histone demethylase JMJD3 (called JMJD3c). We found that transient, forced demethylation of H3K27me3 alone triggers the upregulation of mesoendodermal genes, even when the culture conditions for the hPSCs are not changed. Furthermore, transient and forced expression of JMJD3c followed by the forced expression of lineage-defining transcription factors enabled the hPSCs to activate tissue-specific genes directly. We have also shown that the introduction of JMJD3c facilitates the differentiation of hPSCs into functional hepatic cells and skeletal muscle cells. These results suggest the utility of the direct manipulation of epigenomes for generating desired cell types from hPSCs for cell transplantation therapy and platforms for drug screenings. PMID:27802135

Isolation and Characterization of DkPK Genes Associated with Natural Deastringency in C-PCNA Persimmon

PubMed Central

Chen, Wenxing; Mo, Rongli; Du, Xiaoyun; Zhang, Qinglin; Luo, Zhengrong

2016-01-01

Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) is considered to be an important germplasm resource for the breeding of PCNA cultivars, though its molecular mechanisms of astringency removal remain to be elucidated. Previously, we showed that the abundance of pyruvate kinase gene transcripts increased rapidly during astringency removal in C-PCNA persimmon fruit. Here, we report the full-length coding sequences of six novel DkPK genes from C-PCNA persimmon fruit isolated based on a complementary DNA (cDNA) library and transcriptome data. The expression patterns of these six DkPK genes and correlations with the soluble proanthocyanidin (PA) content were analyzed during various fruit development stages in different types of persimmon, with DkPK1 showing an expression pattern during the last stage in C-PCNA persimmon that was positively correlated with a decrease in soluble PAs. Phylogenetic analysis revealed that DkPK1 belongs to cytosolic-1 subgroup, and subcellular localization analysis confirmed that DkPK1 is located in the cytosol. Notably, tissue expression profiling revealed ubiquitous DkPK1 expression in different persimmon organs, with the highest expression in seeds. Furthermore, transient over-expression of DkPK1 in persimmon leaves resulted in a significant decrease in the content of soluble PAs but a significant increase in the transcript levels of pyruvate decarboxylase genes (DkPDC1, -3, -4, -5), which catalyze the conversion of pyruvate to acetaldehyde. Thus, we propose that an acetaldehyde-based coagulation effect reduces the content of soluble PAs. Taken together, our results suggest that DkPK1 might be involved in the natural removal of astringency at the last developmental stage in C-PCNA persimmon. This is the first report to identify several novel full-length DkPK genes as well as their potential roles in the natural loss of astringency in C-PCNA persimmon. PMID:26925075

Studying Axon-Astrocyte Functional Interactions by 3D Two-Photon Ca2+ Imaging: A Practical Guide to Experiments and "Big Data" Analysis.

PubMed

Savtchouk, Iaroslav; Carriero, Giovanni; Volterra, Andrea

2018-01-01

Recent advances in fast volumetric imaging have enabled rapid generation of large amounts of multi-dimensional functional data. While many computer frameworks exist for data storage and analysis of the multi-gigabyte Ca 2+ imaging experiments in neurons, they are less useful for analyzing Ca 2+ dynamics in astrocytes, where transients do not follow a predictable spatio-temporal distribution pattern. In this manuscript, we provide a detailed protocol and commentary for recording and analyzing three-dimensional (3D) Ca 2+ transients through time in GCaMP6f-expressing astrocytes of adult brain slices in response to axonal stimulation, using our recently developed tools to perform interactive exploration, filtering, and time-correlation analysis of the transients. In addition to the protocol, we release our in-house software tools and discuss parameters pertinent to conducting axonal stimulation/response experiments across various brain regions and conditions. Our software tools are available from the Volterra Lab webpage at https://wwwfbm.unil.ch/dnf/group/glia-an-active-synaptic-partner/member/volterra-andrea-volterra in the form of software plugins for Image J (NIH)-a de facto standard in scientific image analysis. Three programs are available: MultiROI_TZ_profiler for interactive graphing of several movable ROIs simultaneously, Gaussian_Filter5D for Gaussian filtering in several dimensions, and Correlation_Calculator for computing various cross-correlation parameters on voxel collections through time.

Isolation and functional characterization of Lycopene β-cyclase (CYC-B) promoter from Solanum habrochaites

PubMed Central

2010-01-01

Background Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor β-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene β-cyclase (CYC-B) promoter from a green fruited S. habrochaites genotype EC520061. Results A 908 bp region upstream to the initiation codon of the Lycopene β-cyclase gene was cloned and identified as full-length promoter. To identify promoter region necessary for regulating developmental expression of the ShCYC-B gene, the full-length promoter and its three different 5' truncated fragments were cloned upstream to the initiation codon of GUS reporter cDNA in binary vectors. These four plant transformation vectors were separately transformed in to Agrobacterium. Agrobacterium-mediated transient and stable expression systems were used to study the GUS expression driven by the full-length promoter and its 5' deletion fragments in tomato. The full-length promoter showed a basal level activity in leaves, and its expression was upregulated > 5-fold in flowers and fruits in transgenic tomato plants. Deletion of -908 to -577 bp 5' to ATG decreases the ShCYC-B promoter strength, while deletion of -908 to -437 bp 5' to ATG led to significant increase in the activity of GUS in the transgenic plants. Promoter deletion analysis led to the identification of a short promoter region (-436 bp to ATG) that exhibited a higher promoter strength but similar developmental expression pattern as compared with the full-length ShCYC-B promoter. Conclusion Functional characterization of the full-length ShCYC-B promoter and its deletion fragments in transient expression system in fruto as well as in stable transgenic tomato revealed that the promoter is developmentally regulated and its expression is upregulated in chromoplast-rich flowers and fruits. Our study identified a short promoter region with functional activity and developmental expression pattern similar to that of the full-length ShCYC-B promoter. This 436 bp promoter region can be used in promoter::reporter fusion molecular genetic screens to identify mutants impaired in CYC-B expression, and thus can be a valuable tool in understanding carotenoid metabolism in tomato. Moreover, this short promoter region of ShCYC-B may be useful in genetic engineering of carotenoid content and other agronomic traits in tomato fruits. PMID:20380705

Expression Profiling of the Transient Receptor Potential Vanilloid (TRPV) Channels 1, 2, 3 and 4 in Mucosal Epithelium of Human Ulcerative Colitis.

PubMed

Rizopoulos, Theodoros; Papadaki-Petrou, Helen; Assimakopoulou, Martha

2018-06-15

The Transient Receptor Potential (TRP) family of selective and non-selective ion channels is well represented throughout the mammalian gastrointestinal track. Several members of the Transient Receptor Potential Vanilloid (TRPV) subfamily have been identified in contributing to modulation of mobility, secretion and sensitivity of the human intestine. Previous studies have focused on the detection of TRPV mRNA levels in colon tissue of patients with inflammatory bowel disease (IBD) whereas little information exists regarding TRPV channel expression in the colonic epithelium. The aim of this study was to evaluate the expression levels of TRPV1, TRPV2, TRPV3 and TRPV4 in mucosa epithelial cells of colonic biopsies from patients with ulcerative colitis (UC) in comparison to colonic resections from non-IBD patients (control group). Immunohistochemistry, using specific antibodies and quantitative analyses of TRPV-immunostained epithelial cells, was performed in semi-serial sections of the samples. TRPV1 expression was significantly decreased whereas TRPV4 expression was significantly increased in the colonic epithelium of UC patients compared to patients in the control group ( p < 0.05). No significant difference for TRPV2 and TRPV3 expression levels between UC and control specimens was detected ( p > 0.05). There was no correlation between TRPV channel expression and the clinical features of the disease ( p > 0.05). Further investigation is needed to clarify the role of TRPV channels in human bowel inflammatory response.

Inducible Defenses Stay Up Late: Temporal Patterns of Immune Gene Expression in Tenebrio molitor

PubMed Central

Johnston, Paul R; Makarova, Olga; Rolff, Jens

2014-01-01

The course of microbial infection in insects is shaped by a two-stage process of immune defense. Constitutive defenses, such as engulfment and melanization, act immediately and are followed by inducible defenses, archetypically the production of antimicrobial peptides, which eliminate or suppress the remaining microbes. By applying RNAseq across a 7-day time course, we sought to characterize the long-lasting immune response to bacterial challenge in the mealworm beetle Tenebrio molitor, a model for the biochemistry of insect immunity and persistent bacterial infection. By annotating a hybrid de novo assembly of RNAseq data, we were able to identify putative orthologs for the majority of components of the conserved insect immune system. Compared with Tribolium castaneum, the most closely related species with a reference genome sequence and a manually curated immune system annotation, the T. molitor immune gene count was lower, with lineage-specific expansions of genes encoding serine proteases and their countervailing inhibitors accounting for the majority of the deficit. Quantitative mapping of RNAseq reads to the reference assembly showed that expression of genes with predicted functions in cellular immunity, wound healing, melanization, and the production of reactive oxygen species was transiently induced immediately after immune challenge. In contrast, expression of genes encoding antimicrobial peptides or components of the Toll signaling pathway and iron sequestration response remained elevated for at least 7 days. Numerous genes involved in metabolism and nutrient storage were repressed, indicating a possible cost of immune induction. Strikingly, the expression of almost all antibacterial peptides followed the same pattern of long-lasting induction, regardless of their spectra of activity, signaling possible interactive roles in vivo. PMID:24318927

Inducible defenses stay up late: temporal patterns of immune gene expression in Tenebrio molitor.

PubMed

Johnston, Paul R; Makarova, Olga; Rolff, Jens

2013-12-06

The course of microbial infection in insects is shaped by a two-stage process of immune defense. Constitutive defenses, such as engulfment and melanization, act immediately and are followed by inducible defenses, archetypically the production of antimicrobial peptides, which eliminate or suppress the remaining microbes. By applying RNAseq across a 7-day time course, we sought to characterize the long-lasting immune response to bacterial challenge in the mealworm beetle Tenebrio molitor, a model for the biochemistry of insect immunity and persistent bacterial infection. By annotating a hybrid de novo assembly of RNAseq data, we were able to identify putative orthologs for the majority of components of the conserved insect immune system. Compared with Tribolium castaneum, the most closely related species with a reference genome sequence and a manually curated immune system annotation, the T. molitor immune gene count was lower, with lineage-specific expansions of genes encoding serine proteases and their countervailing inhibitors accounting for the majority of the deficit. Quantitative mapping of RNAseq reads to the reference assembly showed that expression of genes with predicted functions in cellular immunity, wound healing, melanization, and the production of reactive oxygen species was transiently induced immediately after immune challenge. In contrast, expression of genes encoding antimicrobial peptides or components of the Toll signaling pathway and iron sequestration response remained elevated for at least 7 days. Numerous genes involved in metabolism and nutrient storage were repressed, indicating a possible cost of immune induction. Strikingly, the expression of almost all antibacterial peptides followed the same pattern of long-lasting induction, regardless of their spectra of activity, signaling possible interactive roles in vivo. Copyright © 2014 Johnston et al.

Imprint switch mutations at Rasgrf1 support conflict hypothesis of imprinting and define a growth control mechanism upstream of IGF1

PubMed Central

Drake, Nadia M.; Park, Yoon Jung; Shirali, Aditya S.; Cleland, Thomas A.

2010-01-01

Rasgrf1 is imprinted and expressed preferentially from the paternal allele in neonatal mouse brain. At weaning, expression becomes biallelic. Using a mouse model, we assayed the effects of perturbing imprinted Rasgrf1 expression in mice with the following imprinted expression patterns: monoallelic paternal (wild type), monoallelic maternal (maternal only), biallelic (both alleles transcribed), and null (neither allele transcribed). All genotypes exhibit biallelic expression around weaning. Consequences of this transient imprinting perturbation are manifested as overall size differences that correspond to the amount of neonatal Rasgrf1 expressed and are persistent, extending into adulthood. Biallelic mice are the largest and overexpress Rasgrf1 relative to wild-type mice, null mice are the smallest and underexpress Rasgrf1 as neonates, and the two monoallelically expressing genotypes are intermediate and indistinguishable from one another, in both size and Rasgrf1 expression level. Importantly, these data support one of the key underlying assumptions of the “conflict hypothesis” that describes the evolution of genomic imprinting in mammals and supposes that equivalent amounts of imprinted gene expression produce equivalent phenotypes, regardless of which parental allele is transcribed. Concordant with the difference in overall body size, we identify differences in IGF-1 levels, both in serum protein and as liver transcript, and identify additional differential expression of components upstream of IGF-1 release in the GH/IGF-1 axis. These data suggest that imprinted Rasgrf1 expression affects GH/IGF-1 axis function, and that the consequences of Rasgrf1 inputs to this axis persist beyond the time period when expression is restricted via epigenetic mechanisms, suggesting that proper neonatal Rasgrf1 expression levels are critical for development. PMID:19513790

Linking suckling biomechanics to the development of the palate

NASA Astrophysics Data System (ADS)

Li, Jingtao; Johnson, Chelsey A.; Smith, Andrew A.; Hunter, Daniel J.; Singh, Gurpreet; Brunski, John B.; Helms, Jill A.

2016-02-01

Skulls are amongst the most informative documents of evolutionary history but a complex geometry, coupled with composite material properties and complicated biomechanics, have made it particularly challenging to identify mechanical principles guiding the skull’s morphogenesis. Despite this challenge, multiple lines of evidence, for example the relationship between masticatory function and the evolution of jaw shape, nonetheless suggest that mechanobiology plays a major role in skull morphogenesis. To begin to tackle this persistent challenge, cellular, molecular and tissue-level analyses of the developing mouse palate were coupled with finite element modeling to demonstrate that patterns of strain created by mammalian-specific oral behaviors produce complementary patterns of chondrogenic gene expression in an initially homogeneous population of cranial neural crest cells. Neural crest cells change from an osteogenic to a chondrogenic fate, leading to the materialization of cartilaginous growth plate-like structures in the palatal midline. These growth plates contribute to lateral expansion of the head but are transient structures; when the strain patterns associated with suckling dissipate at weaning, the growth plates disappear and the palate ossifies. Thus, mechanical cues such as strain appear to co-regulate cell fate specification and ultimately, help drive large-scale morphogenetic changes in head shape.

The role of ZmWRKY4 in regulating maize antioxidant defense under cadmium stress.

PubMed

Hong, Changyong; Cheng, Dan; Zhang, Guoqiang; Zhu, Dandan; Chen, Yahua; Tan, Mingpu

2017-01-22

WRKY transcription factors act as positive regulators in abiotic stress responses by activation of the cellular antioxidant systems. However, there are few reports on the response of WRKY genes to cadmium (Cd) stress. In this study, the role of maize ZmWRKY4 in regulating antioxidant enzymes in Cd stress was investigated. The results indicated that Cd induced up-regulation of the expression and the activities of ZmWRKY4 and superoxide dismutase (SOD) and ascorbate peroxidase (APX). Transient expression and RNA interference (RNAi) silencing of ZmWRKY4 in maize mesophyll protoplasts further revealed that ZmWRKY4 was required for the abscisic acid (ABA)-induced increase in expression and activity of SOD and APX. Overexpression of ZmWRKY4 in protoplasts upregulated the expression and the activities of antioxidant enzymes, whereas ABA induced increases in the expression and the activities of antioxidant enzymes were blocked by the RNAi silencing of ZmWRKY4. Bioinformatic analysis indicated that ZmSOD4 and ZmcAPX both harbored two W-boxes, binding motif for WRKY transcription factors, in their promoter region. Intriguingly, ZmWRKY4 belongs to group I WRKYs with two WRKY domains. Moreover, the synchronized expression patterns indicate that ZmWRKY4 might play a critical role in either regulating the ZmSOD4 and ZmcAPX expression or cooperating with them in response to stress and phytohormone. Copyright © 2016 Elsevier Inc. All rights reserved.

Cluster analysis of multiple planetary flow regimes

NASA Technical Reports Server (NTRS)

Mo, Kingtse; Ghil, Michael

1987-01-01

A modified cluster analysis method was developed to identify spatial patterns of planetary flow regimes, and to study transitions between them. This method was applied first to a simple deterministic model and second to Northern Hemisphere (NH) 500 mb data. The dynamical model is governed by the fully-nonlinear, equivalent-barotropic vorticity equation on the sphere. Clusters of point in the model's phase space are associated with either a few persistent or with many transient events. Two stationary clusters have patterns similar to unstable stationary model solutions, zonal, or blocked. Transient clusters of wave trains serve as way stations between the stationary ones. For the NH data, cluster analysis was performed in the subspace of the first seven empirical orthogonal functions (EOFs). Stationary clusters are found in the low-frequency band of more than 10 days, and transient clusters in the bandpass frequency window between 2.5 and 6 days. In the low-frequency band three pairs of clusters determine, respectively, EOFs 1, 2, and 3. They exhibit well-known regional features, such as blocking, the Pacific/North American (PNA) pattern and wave trains. Both model and low-pass data show strong bimodality. Clusters in the bandpass window show wave-train patterns in the two jet exit regions. They are related, as in the model, to transitions between stationary clusters.

Suppression of transient enhanced diffusion in sub-micron patterned silicon template by dislocation loops formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Kuan-Kan; Woon, Wei Yen; Chang, Ruey-Dar

We investigate the evolution of two dimensional transient enhanced diffusion (TED) of phosphorus in sub-micron scale patterned silicon template. Samples doped with low dose phosphorus with and without high dose silicon self-implantation, were annealed for various durations. Dopant diffusion is probed with plane-view scanning capacitance microscopy. The measurement revealed two phases of TED. Significant suppression in the second phase TED is observed for samples with high dose self-implantation. Transmission electron microscopy suggests the suppressed TED is related to the evolution of end of range defect formed around ion implantation sidewalls.

Suppression of transient enhanced diffusion in sub-micron patterned silicon template by dislocation loops formation

NASA Astrophysics Data System (ADS)

Hu, Kuan-Kan; Chang, Ruey-Dar; Woon, Wei Yen

2015-10-01

We investigate the evolution of two dimensional transient enhanced diffusion (TED) of phosphorus in sub-micron scale patterned silicon template. Samples doped with low dose phosphorus with and without high dose silicon self-implantation, were annealed for various durations. Dopant diffusion is probed with plane-view scanning capacitance microscopy. The measurement revealed two phases of TED. Significant suppression in the second phase TED is observed for samples with high dose self-implantation. Transmission electron microscopy suggests the suppressed TED is related to the evolution of end of range defect formed around ion implantation sidewalls.

Increased avidity for Dpp/BMP2 maintains the proliferation of progenitors-like cells in the Drosophila eye.

PubMed

Neto, Marta; Aguilar-Hidalgo, Daniel; Casares, Fernando

2016-10-01

During organ development, the progenitor state is transient, and depends on specific combinations of transcription factors and extracellular signals. Not surprisingly, abnormal maintenance of progenitor transcription factors may lead to tissue overgrowth, and the concurrence of signals from the local environment is often critical to trigger this overgrowth. Therefore, identifying specific combinations of transcription factors/signals promoting -or opposing- proliferation in progenitors is essential to understand normal development and disease. We have investigated this issue using the Drosophila eye as model. Transcription factors hth and tsh are transiently expressed in eye progenitors causing the expansion of the progenitor pool. However, if their co-expression is maintained experimentally, cell proliferation continues and differentiation is halted. Here we show that Hth+Tsh-induced tissue overgrowth requires the BMP2 Dpp and the abnormal hyperactivation of its pathway. Rather than using autocrine Dpp expression, Hth+Tsh cells increase their avidity for Dpp, produced locally, by upregulating extracellular matrix components. During normal development, Dpp represses hth and tsh ensuring that the progenitor state is transient. However, cells in which Hth+Tsh expression is forcibly maintained use Dpp to enhance their proliferation. Copyright © 2016 Elsevier Inc. All rights reserved.

High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

NASA Technical Reports Server (NTRS)

Wenck, A. R.; Quinn, M.; Whetten, R. W.; Pullman, G.; Sederoff, R.; Brown, C. S. (Principal Investigator)

1999-01-01

Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

Functional Characterization of a Bidirectional Plant Promoter from Cotton Leaf Curl Burewala Virus Using an Agrobacterium-Mediated Transient Assay

PubMed Central

Ashraf, Muhammad Aleem; Shahid, Ahmad Ali; Rao, Abdul Qayyum; Bajwa, Kamran Shehzad; Husnain, Tayyab

2014-01-01

The C1 promoter expressing the AC1 gene, and V1 promoter expressing the AV1 gene are located in opposite orientations in the large intergenic region of the Cotton leaf curl Burewala virus (CLCuBuV) genome. Agro-infiltration was used to transiently express putative promoter constructs in Nicotiana tabacum and Gossypium hirsutum leaves, which was monitored by a GUS reporter gene, and revealed that the bidirectional promoter of CLCuBuV transcriptionally regulates both the AC1 and AV1 genes. The CLCuBuV C1 gene promoter showed a strong, consistent transient expression of the reporter gene (GUS) in N. tabacum and G. hirsutum leaves and exhibited GUS activity two- to three-fold higher than the CaMV 35S promoter. The CLCuBuV bidirectional genepromoter is a nearly constitutive promoter that contains basic conserved elements. Many cis-regulatory elements (CREs) were also analyzed within the bidirectional plant promoters of CLCuBuV and closely related geminiviruses, which may be helpful in understanding the transcriptional regulation of both the virus and host plant. PMID:24424501

Integrins engage mitochondrial function for signal transduction by a mechanism dependent on Rho GTPases

PubMed Central

Werner, Erica; Werb, Zena

2002-01-01

We show here the transient activation of the small GTPase Rac, followed by a rise in reactive oxygen species (ROS), as necessary early steps in a signal transduction cascade that lead to NFκB activation and collagenase-1 (CL-1)/matrix metalloproteinase-1 production after integrin-mediated cell shape changes. We show evidence indicating that this constitutes a new mechanism for ROS production mediated by small GTPases. Activated RhoA also induced ROS production and up-regulated CL-1 expression. A Rac mutant (L37) that prevents reorganization of the actin cytoskeleton prevented integrin-induced CL-1 expression, whereas mutations that abrogate Rac binding to the neutrophil NADPH membrane oxidase in vitro (H26 and N130) did not. Instead, ROS were produced by integrin-induced changes in mitochondrial function, which were inhibited by Bcl-2 and involved transient membrane potential loss. The cells showing this transient decrease in mitochondrial membrane potential were already committed to CL-1 expression. These results unveil a new molecular mechanism of signal transduction triggered by integrin engagement where a global mitochondrial metabolic response leads to gene expression rather than apoptosis. PMID:12119354

Integrins engage mitochondrial function for signal transduction by a mechanism dependent on Rho GTPases.

PubMed

Werner, Erica; Werb, Zena

2002-07-22

We show here the transient activation of the small GTPase Rac, followed by a rise in reactive oxygen species (ROS), as necessary early steps in a signal transduction cascade that lead to NFkappaB activation and collagenase-1 (CL-1)/matrix metalloproteinase-1 production after integrin-mediated cell shape changes. We show evidence indicating that this constitutes a new mechanism for ROS production mediated by small GTPases. Activated RhoA also induced ROS production and up-regulated CL-1 expression. A Rac mutant (L37) that prevents reorganization of the actin cytoskeleton prevented integrin-induced CL-1 expression, whereas mutations that abrogate Rac binding to the neutrophil NADPH membrane oxidase in vitro (H26 and N130) did not. Instead, ROS were produced by integrin-induced changes in mitochondrial function, which were inhibited by Bcl-2 and involved transient membrane potential loss. The cells showing this transient decrease in mitochondrial membrane potential were already committed to CL-1 expression. These results unveil a new molecular mechanism of signal transduction triggered by integrin engagement where a global mitochondrial metabolic response leads to gene expression rather than apoptosis.

Transient gestational and neonatal hypothyroidism-induced specific changes in androgen receptor expression in skeletal and cardiac muscles of adult rat.

PubMed

Annapoorna, K; Anbalagan, J; Neelamohan, R; Vengatesh, G; Stanley, J; Amudha, G; Aruldhas, M M

2013-03-01

The present study aims to identify the association between androgen status and metabolic activity in skeletal and cardiac muscles of adult rats with transient gestational/neonatal-onset hypothyroidism. Pregnant and lactating rats were made hypothyroid by exposing to 0.05% methimazole in drinking water; gestational exposure was from embryonic day 9-14 (group II) or 21 (group III), lactational exposure was from postnatal day 1-14 (group IV) or 29 (group V). Serum was collected for hormone assay. Androgen receptor status, Glu-4 expression, and enzyme activities were assessed in the skeletal and cardiac muscles. Serum testosterone and estradiol levels decreased in adult rats of groups II and III, whereas testosterone remained normal but estradiol increased in group IV and V, when compared to coeval control. Androgen receptor ligand binding activity increased in both muscle phenotypes with a consistent increase in the expression level of its mRNA and protein expressions except in the forelimb of adult rats with transient hypothyroidism (group II-V). Glut-4 expression remained normal in skeletal and cardiac muscle of experimental rats. Specific activity of hexokinase and lactate dehydrogenase increased in both muscle phenotypes whereas, creatine kinase activity increased in skeletal muscles alone. It is concluded that transient gestational/lactational exposure to methimazole results in hypothyroidism during prepuberal life whereas it increases AR status and glycolytic activity in skeletal and cardiac muscles even at adulthood. Thus, the present study suggests that euthyroid status during prenatal and early postnatal life is essential to have optimal AR status and metabolic activity at adulthood. © Georg Thieme Verlag KG Stuttgart · New York.

Transient transcriptional activation of the Vibrio cholerae El Tor virulence regulator toxT in response to culture conditions.

PubMed

Medrano, A I; DiRita, V J; Castillo, G; Sanchez, J

1999-05-01

Vibrio cholerae El Tor require special in vitro culture conditions, consisting of an initial static growth period followed by shift to shaking (AKI conditions), for expression of cholera toxin (CT) and toxin coregulated pili (TCP). ToxT, a regulator whose initial transcription depends on the ToxR regulator, positively modulates expression of CT and TCP. To help understand control of CT and TCP in El Tor vibrios, we monitored ctxAB and ToxR-dependent toxT transcription by time course primer extension assays. AKI conditions stimulated CT synthesis with an absence of ctxAB transcription during static growth followed by induction upon shaking. ToxR-dependent toxT transcription was induced at the end of the static growth period but was transient, stopping shortly after shaking was initiated but, interestingly, also if the static phase was prolonged. Immunoblot assays showed that ToxR protein levels were not coincidentally transient, implying a protein on/off switch mechanism for ToxR. Despite the transient activation by ToxR, transcription of ctxAB was maintained during shaking. This finding suggested continued toxT expression, possibly through relay transcription from another promoter. The 12.6-kb distant upstream tcpA promoter responsible for expression of the TCP operon has been proposed to provide an alternate toxT message by readthrough transcription. Activation of the tcpA promoter is supported by increased expression of TcpA protein during the shaking phase of the culture. Readthrough transcription of toxT from tcpA would be compatible with reverse transcription-PCR evidence for a toxT mRNA at times when ToxR-dependent transcription was no longer detectable by primer extension.

Animal component-free Agrobacterium tumefaciens cultivation media for better GMP-compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana.

PubMed

Houdelet, Marcel; Galinski, Anna; Holland, Tanja; Wenzel, Kathrin; Schillberg, Stefan; Buyel, Johannes Felix

2017-04-01

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large-scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal-derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal-derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design-of-experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two-fold increase in OD 600 compared to YEB medium during a 4-L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal-derived components, thus facilitating the GMP-compliant large-scale transient expression of recombinant proteins in plants. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamic Expression of the Translational Machinery during Bacillus subtilis Life Cycle at a Single Cell Level

PubMed Central

Rosenberg, Alex; Sinai, Lior; Smith, Yoav; Ben-Yehuda, Sigal

2012-01-01

The ability of bacteria to responsively regulate the expression of translation components is crucial for rapid adaptation to fluctuating environments. Utilizing Bacillus subtilis (B. subtilis) as a model organism, we followed the dynamics of the translational machinery at a single cell resolution during growth and differentiation. By comprehensive monitoring the activity of the major rrn promoters and ribosomal protein production, we revealed diverse dynamics between cells grown in rich and poor medium, with the most prominent dissimilarities exhibited during deep stationary phase. Further, the variability pattern of translational activity varied among the cells, being affected by nutrient availability. We have monitored for the first time translational dynamics during the developmental process of sporulation within the two distinct cellular compartments of forespore and mother-cell. Our study uncovers a transient forespore specific increase in expression of translational components. Finally, the contribution of each rrn promoter throughout the bacterium life cycle was found to be relatively constant, implying that differential expression is not the main purpose for the existence of multiple rrn genes. Instead, we propose that coordination of the rrn operons serves as a strategy to rapidly fine tune translational activities in a synchronized fashion to achieve an optimal translation level for a given condition. PMID:22848659

Transient expression and cellular localization of recombinant proteins in cultured insect cells

USDA-ARS?s Scientific Manuscript database

Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

Partial migration and transient coexistence of migrants and residents in animal populations.

PubMed

Singh, Navinder J; Leonardsson, Kjell

2014-01-01

Partial migration, whereby a proportion of the population migrates, is common across the animal kingdom. Much of the focus in the literature has been on trying to explain the underlying mechanisms for the coexistence of migrants and residents. In addition, there has been an increasing number of reports on the prevalence and frequency of partially migratory populations. One possible explanation for the occurrence of partial migration, which has received no attention in the literature, is that of 'transient coexistence' during the invasion phase of a superior behaviour. In this study we develop a theoretical basis for explaining partial migration as a transient coexistence and derive a method to predict the frequency of residents and migrants in partially migrating populations. This method is useful to predict the frequencies of migrants and residents in a small set of populations as a complementing hypothesis to 'an Evolutionary Stable Strategy (ESS)'. We use the logistic growth equation to derive a formula for predicting the frequencies of residents and migrants. We also use simulations and empirical data from white perch (Morone americana), moose (Alces alces) and red deer (Cervus elaphus) to demonstrate our approach. We show that the probability of detecting partial migration due to transient coexistence depends upon a minimum number of tracked or marked individuals for a given number of populations. Our approach provides a starting point in searching for explanations to the observed frequencies, by contrasting the observed pattern with both the predicted transient and the uniform random pattern. Aggregating such information on observed patterns (proportions of migrants and residents) may eventually lead to the development of a quantitative theory for the equilibrium (ESS) populations as well.

Identification and Expression Profiling of the Auxin Response Factors in Dendrobium officinale under Abiotic Stresses

PubMed Central

Chen, Zhehao; Yuan, Ye; Fu, Di; Shen, Chenjia; Yang, Yanjun

2017-01-01

Auxin response factor (ARF) proteins play roles in plant responses to diverse environmental stresses by binding specifically to the auxin response element in the promoters of target genes. Using our latest public Dendrobium transcriptomes, a comprehensive characterization and analysis of 14 DnARF genes were performed. Three selected DnARFs, including DnARF1, DnARF4, and DnARF6, were confirmed to be nuclear proteins according to their transient expression in epidermal cells of Nicotiana benthamiana leaves. Furthermore, the transcription activation abilities of DnARF1, DnARF4, and DnARF6 were tested in a yeast system. Our data showed that DnARF6 is a transcriptional activator in Dendrobium officinale. To uncover the basic information of DnARF gene responses to abiotic stresses, we analyzed their expression patterns under various hormones and abiotic treatments. Based on our data, several hormones and significant stress responsive DnARF genes have been identified. Since auxin and ARF genes have been identified in many plant species, our data is imperative to reveal the function of ARF mediated auxin signaling in the adaptation to the challenging Dendrobium environment. PMID:28471373

Identification and Expression Profiling of the Auxin Response Factors in Dendrobium officinale under Abiotic Stresses.

PubMed

Chen, Zhehao; Yuan, Ye; Fu, Di; Shen, Chenjia; Yang, Yanjun

2017-05-04

Auxin response factor (ARF) proteins play roles in plant responses to diverse environmental stresses by binding specifically to the auxin response element in the promoters of target genes. Using our latest public Dendrobium transcriptomes, a comprehensive characterization and analysis of 14 DnARF genes were performed. Three selected DnARFs , including DnARF1 , DnARF4 , and DnARF6 , were confirmed to be nuclear proteins according to their transient expression in epidermal cells of Nicotiana benthamiana leaves. Furthermore, the transcription activation abilities of DnARF1 , DnARF4 , and DnARF6 were tested in a yeast system. Our data showed that DnARF6 is a transcriptional activator in Dendrobium officinale . To uncover the basic information of DnARF gene responses to abiotic stresses, we analyzed their expression patterns under various hormones and abiotic treatments. Based on our data, several hormones and significant stress responsive DnARF genes have been identified. Since auxin and ARF genes have been identified in many plant species, our data is imperative to reveal the function of ARF mediated auxin signaling in the adaptation to the challenging Dendrobium environment.

Rapid Software-Based Design and Optical Transient Liquid Molding of Microparticles.

PubMed

Wu, Chueh-Yu; Owsley, Keegan; Di Carlo, Dino

2015-12-22

Microparticles with complex 3D shape and composition are produced using a novel fabrication method, optical transient liquid molding, in which a 2D light pattern exposes a photopolymer precursor stream shaped along the flow axis by software-aided inertial flow engineering. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies.

PubMed

Chung, Nancy P Y; Matthews, Katie; Kim, Helen J; Ketas, Thomas J; Golabek, Michael; de Los Reyes, Kevin; Korzun, Jacob; Yasmeen, Anila; Sanders, Rogier W; Klasse, Per Johan; Wilson, Ian A; Ward, Andrew B; Marozsan, Andre J; Moore, John P; Cupo, Albert

2014-04-25

Recombinant soluble, cleaved HIV-1 envelope glycoprotein SOSIP.664 gp140 trimers based on the subtype A BG505 sequence are being studied structurally and tested as immunogens in animals. For these trimers to become a vaccine candidate for human trials, they would need to be made in appropriate amounts at an acceptable quality. Accomplishing such tasks by transient transfection is likely to be challenging. The traditional way to express recombinant proteins in large amounts is via a permanent cell line, usually of mammalian origin. Making cell lines that produce BG505 SOSIP.664 trimers requires the co-expression of the Furin protease to ensure that the cleavage site between the gp120 and gp41 subunits is fully utilized. We designed a vector capable of expressing Env and Furin, and used it to create Stable 293 T and CHO Flp-In™ cell lines through site-specific recombination. Both lines produce high quality, cleaved trimers at yields of up to 12-15 mg per 1 × 109 cells. Trimer expression at such levels was maintained for up to 30 days (10 passages) after initial seeding and was consistently superior to what could be achieved by transient transfection. Electron microscopy studies confirm that the purified trimers have the same native-like appearance as those derived by transient transfection and used to generate high-resolution structures. They also have appropriate antigenic properties, including the presentation of the quaternary epitope for the broadly neutralizing antibody PGT145. The BG505 SOSIP.664 trimer-expressing cell lines yield proteins of an appropriate quality for structural studies and animal immunogenicity experiments. The methodology is suitable for making similar lines under Good Manufacturing Practice conditions, to produce trimers for human clinical trials. Moreover, any env gene can be incorporated into this vector system, allowing the manufacture of SOSIP trimers from multiple genotypes, either by transient transfection or from stable cell lines.

Transient radiation in an anisotropic magnetodielectric plate in a waveguide

NASA Astrophysics Data System (ADS)

Gevorkyan, E. A.

2017-02-01

We have considered transient radiation of a charged particle in an anisotropic magnetodielectric plate placed into a regular waveguide. It is assumed that the charged particle passes through the plate moving at a constant velocity perpendicularly to the waveguide axis. Wave equations and analytical expressions for transverse electric (TE) and transverse magnetic (TM) fields in different regions of the waveguide have been obtained. Energies of transient radiation of the moving particle have been calculated. The properties of transient radiation and Vavilov-Cherenkov radiation have been analyzed for the case of a rectangular waveguide. Energies of transient radiation have been calculated for the case of a "thin" plate in the waveguide, when the wavelength in the plate is much greater than the length of the plate.

Patterning of functional human astrocytes onto parylene-C/SiO2 substrates for the study of Ca2+ dynamics in astrocytic networks

NASA Astrophysics Data System (ADS)

Raos, B. J.; Simpson, M. C.; Doyle, C. S.; Murray, A. F.; Graham, E. S.; Unsworth, C. P.

2018-06-01

Objective. Recent literature suggests that astrocytes form organized functional networks and communicate through transient changes in cytosolic Ca2+. Traditional techniques to investigate network activity, such as pharmacological blocking or genetic knockout, are difficult to restrict to individual cells. The objective of this work is to develop cell-patterning techniques to physically manipulate astrocytic interactions to enable the study of Ca2+ in astrocytic networks. Approach. We investigate how an in vitro cell-patterning platform that utilizes geometric patterns of parylene-C on SiO2 can be used to physically isolate single astrocytes and small astrocytic networks. Main results. We report that single astrocytes are effectively isolated on 75  ×  75 µm square parylene nodes, whereas multi-cellular astrocytic networks are isolated on larger nodes, with the mean number of astrocytes per cluster increasing as a function of node size. Additionally, we report that astrocytes in small multi-cellular clusters exhibit spatio-temporal clustering of Ca2+ transients. Finally, we report that the frequency and regularity of Ca2+ transients was positively correlated with astrocyte connectivity. Significance. The significance of this work is to demonstrate how patterning hNT astrocytes replicates spatio-temporal clustering of Ca2+ signalling that is observed in vivo but not in dissociated in vitro cultures. We therefore highlight the importance of the structure of astrocytic networks in determining ensemble Ca2+ behaviour.

Transcriptomic and metabolite analyses of Cabernet Sauvignon grape berry development.

PubMed

Deluc, Laurent G; Grimplet, Jérôme; Wheatley, Matthew D; Tillett, Richard L; Quilici, David R; Osborne, Craig; Schooley, David A; Schlauch, Karen A; Cushman, John C; Cramer, Grant R

2007-11-22

Grape berry development is a dynamic process that involves a complex series of molecular genetic and biochemical changes divided into three major phases. During initial berry growth (Phase I), berry size increases along a sigmoidal growth curve due to cell division and subsequent cell expansion, and organic acids (mainly malate and tartrate), tannins, and hydroxycinnamates accumulate to peak levels. The second major phase (Phase II) is defined as a lag phase in which cell expansion ceases and sugars begin to accumulate. Véraison (the onset of ripening) marks the beginning of the third major phase (Phase III) in which berries undergo a second period of sigmoidal growth due to additional mesocarp cell expansion, accumulation of anthocyanin pigments for berry color, accumulation of volatile compounds for aroma, softening, peak accumulation of sugars (mainly glucose and fructose), and a decline in organic acid accumulation. In order to understand the transcriptional network responsible for controlling berry development, mRNA expression profiling was conducted on berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip Vitis oligonucleotide microarray ver. 1.0 spanning seven stages of berry development from small pea size berries (E-L stages 31 to 33 as defined by the modified E-L system), through véraison (E-L stages 34 and 35), to mature berries (E-L stages 36 and 38). Selected metabolites were profiled in parallel with mRNA expression profiling to understand the effect of transcriptional regulatory processes on specific metabolite production that ultimately influence the organoleptic properties of wine. Over the course of berry development whole fruit tissues were found to express an average of 74.5% of probes represented on the Vitis microarray, which has 14,470 Unigenes. Approximately 60% of the expressed transcripts were differentially expressed between at least two out of the seven stages of berry development (28% of transcripts, 4,151 Unigenes, had pronounced (> or =2 fold) differences in mRNA expression) illustrating the dynamic nature of the developmental process. The subset of 4,151 Unigenes was split into twenty well-correlated expression profiles. Expression profile patterns included those with declining or increasing mRNA expression over the course of berry development as well as transient peak or trough patterns across various developmental stages as defined by the modified E-L system. These detailed surveys revealed the expression patterns for genes that play key functional roles in phytohormone biosynthesis and response, calcium sequestration, transport and signaling, cell wall metabolism mediating expansion, ripening, and softening, flavonoid metabolism and transport, organic and amino acid metabolism, hexose sugar and triose phosphate metabolism and transport, starch metabolism, photosynthesis, circadian cycles and pathogen resistance. In particular, mRNA expression patterns of transcription factors, abscisic acid (ABA) biosynthesis, and calcium signaling genes identified candidate factors likely to participate in the progression of key developmental events such as véraison and potential candidate genes associated with such processes as auxin partitioning within berry cells, aroma compound production, and pathway regulation and sequestration of flavonoid compounds. Finally, analysis of sugar metabolism gene expression patterns indicated the existence of an alternative pathway for glucose and triose phosphate production that is invoked from véraison to mature berries. These results reveal the first high-resolution picture of the transcriptome dynamics that occur during seven stages of grape berry development. This work also establishes an extensive catalog of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern berry development in a widely grown cultivar of wine grape. More importantly, this analysis identified a set of previously unknown genes potentially involved in critical steps associated with fruit development that can now be subjected to functional testing.

Transcriptomic and metabolite analyses of Cabernet Sauvignon grape berry development

PubMed Central

Deluc, Laurent G; Grimplet, Jérôme; Wheatley, Matthew D; Tillett, Richard L; Quilici, David R; Osborne, Craig; Schooley, David A; Schlauch, Karen A; Cushman, John C; Cramer, Grant R

2007-01-01

Background Grape berry development is a dynamic process that involves a complex series of molecular genetic and biochemical changes divided into three major phases. During initial berry growth (Phase I), berry size increases along a sigmoidal growth curve due to cell division and subsequent cell expansion, and organic acids (mainly malate and tartrate), tannins, and hydroxycinnamates accumulate to peak levels. The second major phase (Phase II) is defined as a lag phase in which cell expansion ceases and sugars begin to accumulate. Véraison (the onset of ripening) marks the beginning of the third major phase (Phase III) in which berries undergo a second period of sigmoidal growth due to additional mesocarp cell expansion, accumulation of anthocyanin pigments for berry color, accumulation of volatile compounds for aroma, softening, peak accumulation of sugars (mainly glucose and fructose), and a decline in organic acid accumulation. In order to understand the transcriptional network responsible for controlling berry development, mRNA expression profiling was conducted on berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0 spanning seven stages of berry development from small pea size berries (E-L stages 31 to 33 as defined by the modified E-L system), through véraison (E-L stages 34 and 35), to mature berries (E-L stages 36 and 38). Selected metabolites were profiled in parallel with mRNA expression profiling to understand the effect of transcriptional regulatory processes on specific metabolite production that ultimately influence the organoleptic properties of wine. Results Over the course of berry development whole fruit tissues were found to express an average of 74.5% of probes represented on the Vitis microarray, which has 14,470 Unigenes. Approximately 60% of the expressed transcripts were differentially expressed between at least two out of the seven stages of berry development (28% of transcripts, 4,151 Unigenes, had pronounced (≥2 fold) differences in mRNA expression) illustrating the dynamic nature of the developmental process. The subset of 4,151 Unigenes was split into twenty well-correlated expression profiles. Expression profile patterns included those with declining or increasing mRNA expression over the course of berry development as well as transient peak or trough patterns across various developmental stages as defined by the modified E-L system. These detailed surveys revealed the expression patterns for genes that play key functional roles in phytohormone biosynthesis and response, calcium sequestration, transport and signaling, cell wall metabolism mediating expansion, ripening, and softening, flavonoid metabolism and transport, organic and amino acid metabolism, hexose sugar and triose phosphate metabolism and transport, starch metabolism, photosynthesis, circadian cycles and pathogen resistance. In particular, mRNA expression patterns of transcription factors, abscisic acid (ABA) biosynthesis, and calcium signaling genes identified candidate factors likely to participate in the progression of key developmental events such as véraison and potential candidate genes associated with such processes as auxin partitioning within berry cells, aroma compound production, and pathway regulation and sequestration of flavonoid compounds. Finally, analysis of sugar metabolism gene expression patterns indicated the existence of an alternative pathway for glucose and triose phosphate production that is invoked from véraison to mature berries. Conclusion These results reveal the first high-resolution picture of the transcriptome dynamics that occur during seven stages of grape berry development. This work also establishes an extensive catalog of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern berry development in a widely grown cultivar of wine grape. More importantly, this analysis identified a set of previously unknown genes potentially involved in critical steps associated with fruit development that can now be subjected to functional testing. PMID:18034876

Differential ontogenetic patterns of levocabastine-sensitive neurotensin NT2 receptors and of NT1 receptors in the rat brain revealed by in situ hybridization.

PubMed

Lépée-Lorgeoux, I; Betancur, C; Rostène, W; Pélaprat, D

1999-03-12

The postnatal ontogeny of the levocabastine-sensitive neurotensin receptor (NT2) mRNA was studied by in situ hybridization in the rat brain and compared with the distribution of the levocabastine-insensitive NT1 receptor. NT2 receptor mRNA was absent at birth from all brain structures except the ependymal cell layer lining the ventricles. The development of NT2 receptor mRNA followed three ontogenetic patterns. The first pattern, involving the majority of the cerebral gray matter, was characterized by a continuous increase from postnatal day 5 (P5) to P30. The second one, involving regions rich in myelinated fibers such as the corpus callosum and lacunosum moleculare layer of the hippocampus, exhibited a pronounced increase between P5 and P10, peaked at P15 and was followed by a plateau or a slight decrease. The third pattern was observed in the ependymal cell layer lining the olfactory and lateral ventricles, where the high labeling already present at birth continued to increase during development. These different developmental patterns could reflect the variety of cells expressing NT2 receptor mRNA, including neurons, protoplasmic astrocytes in gray matter, fibrous astrocytes present in myelinated fibers tracts, and ependymal cells. In contrast, NT1 receptor mRNA, which seems to be associated only with neurons, was highly and transiently expressed during the perinatal period in the cerebral cortex, hippocampus and striatal neuroepithelium. Other regions, notably the ventral tegmental area and substantia nigra compacta, exhibited a gradual increase in NT1 receptor signal, reaching adult levels by P21. Both the differential localization and ontogenetic profiles of NT1 and NT2 receptor mRNAs suggest different involvement of these two receptors in brain functions and development. Copyright 1999 Elsevier Science B.V.

Transient Receptor Potential Ion Channels Control Thermoregulatory Behaviour in Reptiles

PubMed Central

Seebacher, Frank; Murray, Shauna A.

2007-01-01

Biological functions are governed by thermodynamics, and animals regulate their body temperature to optimise cellular performance and to avoid harmful extremes. The capacity to sense environmental and internal temperatures is a prerequisite for the evolution of thermoregulation. However, the mechanisms that enable ectothermic vertebrates to sense heat remain unknown. The recently discovered thermal characteristics of transient receptor potential ion channels (TRP) render these proteins suitable to act as temperature sensors. Here we test the hypothesis that TRPs are present in reptiles and function to control thermoregulatory behaviour. We show that the hot-sensing TRPV1 is expressed in a crocodile (Crocodylus porosus), an agamid (Amphibolurus muricatus) and a scincid (Pseudemoia entrecasteauxii) lizard, as well as in the quail and zebrafinch (Coturnix chinensis and Poephila guttata). The TRPV1 genes from all reptiles form a unique clade that is delineated from the mammalian and the ancestral Xenopus sequences by an insertion of two amino acids. TRPV1 and the cool-sensing TRPM8 are expressed in liver, muscle (transversospinalis complex), and heart tissues of the crocodile, and have the potential to act as internal thermometer and as external temperatures sensors. Inhibition of TRPV1 and TRPM8 in C. porosus abolishes the typically reptilian shuttling behaviour between cooling and heating environments, and leads to significantly altered body temperature patterns. Our results provide the proximate mechanism of thermal selection in terrestrial ectotherms, which heralds a fundamental change in interpretation, because TRPs provide the mechanism for a tissue-specific input into the animals' thermoregulatory response. PMID:17356692

Role of promoter element in c-mpl gene expression induced by TPO.

PubMed

Sunohara, Masataka; Morikawa, Shigeru; Fuse, Akira; Sato, Iwao

2013-01-01

Thrombopoietin (TPO) and its receptor, c-Mpl, play the crucial role for the development of megakaryocyte and considered to regulate megakaryocytopoiesis. Previously we reported that TPO increased the c-mpl promoter activity determined by a transient expression system using a vector containing the luciferase gene as a reporter and the expression of the c-mpl gene is modulated by transcription through a protein kinase C (PKC)-dependent pathway in the megakaryoblastic cells. In this research, to elucidate the required elements in c-mpl promoter, the promoter activity of the deletion constructs and site-directed mutagenesis were measured by a transient transfection assay system. Destruction of -77GATA in c-mpl promoter decreased the activity by 22.8%. Our study elucidated that -77GATA involved in TPO-induced c-mpl gene expression in a human megakaryoblastic cell line, CMK.

Msx genes are expressed in the carapacial ridge of turtle shell: a study of the European pond turtle, Emys orbicularis.

PubMed

Vincent, Christine; Bontoux, Martine; Le Douarin, Nicole M; Pieau, Claude; Monsoro-Burq, Anne-Hélène

2003-09-01

The turtle shell forms by extensive ossification of dermis ventrally and dorsally. The carapacial ridge (CR) controls early dorsal shell formation and is thought to play a similar role in shell growth as the apical ectodermal ridge during limb development. However, the molecular mechanisms underlying carapace development are still unknown. Msx genes are involved in the development of limb mesenchyme and of various skeletal structures. In particular, precocious Msx expression is recorded in skeletal precursors that develop close to the ectoderm, such as vertebral spinous processes or skull. Here, we have studied the embryonic expression of Msx genes in the European pond turtle, Emys orbicularis. The overall Msx expression in head, limb, and trunk is similar to what is observed in other vertebrates. We have focused on the CR area and pre-skeletal shell condensations. The CR expresses Msx genes transiently, in a pattern similar to that of fgf10. In the future carapace domain, the dermis located dorsal to the spinal cord expresses Msx genes, as in other vertebrates, but we did not see expansion of this expression in the dermis located more laterally, on top of the dermomyotomes. In the ventral plastron, although the dermal osseous condensations form in the embryonic Msx-positive somatopleura, we did not observe enhanced Msx expression around these elements. These observations may indicate that common mechanisms participate in limb bud and CR early development, but that pre-differentiation steps differ between shell and other skeletal structures and involve other gene activities than that of Msx genes.

RANKL-induced TRPV2 expression regulates osteoclastogenesis via calcium oscillations.

PubMed

Kajiya, Hiroshi; Okamoto, Fujio; Nemoto, Tetsuomi; Kimachi, Keiichiro; Toh-Goto, Kazuko; Nakayana, Shuji; Okabe, Koji

2010-11-01

The receptor activator of NFκB ligand (RANKL) induces Ca(2+) oscillations and activates the Nuclear Factor of Activated T cells 1 (NFATc1) during osteoclast differentiation (osteoclastogenesis). Ca(2+) oscillations are an important trigger signal for osteoclastogenesis, however the molecular basis of Ca(2+) permeable influx pathways serving Ca(2+) oscillations has not yet been identified. Using a DNA microarray, we found that Transient Receptor Potential Vanilloid channels 2 (TRPV2) are expressed significantly in RANKL-treated RAW264.7 cells (preosteoclasts) compared to untreated cells. Therefore, we further investigated the expression and functional role of TRPV2 on Ca(2+) oscillations and osteoclastogenesis. We found that RANKL dominantly up-regulates TRPV2 expression in preosteoclasts, and evokes spontaneous Ca(2+) oscillations and a transient inward cation current in a time-dependent manner. TRPV inhibitor ruthenium red and tetracycline-induced TRPV2 silencing significantly decreased both the frequency of Ca(2+) oscillations and the transient inward currents in RANKL-treated preosteoclasts. Silencing of store-operated Ca(2+) entry (SOCE) proteins similarly suppressed both RANKL-induced oscillations and currents in preosteoclasts. Furthermore, suppression of TRPV2 also reduced RANKL-induced NAFTc1 expression, its nuclear translocation, and osteoclastogenesis. In summary, Ca(2+) oscillations in preosteoclasts are triggered by RANKL-dependent TRPV2 and SOCE activation and intracellular Ca(2+) release. Subsequent activation of NFATc1 promotes osteoclastogenesis. Copyright © 2010 Elsevier Ltd. All rights reserved.

Expression of Wheat High Molecular Weight Glutenin Subunit 1Bx Is Affected by Large Insertions and Deletions Located in the Upstream Flanking Sequences

PubMed Central

Hao, Chenyang; Tang, Saijun; Zhang, Xueyong; Li, Tian

2014-01-01

To better understand the transcriptional regulation of high molecular weight glutenin subunit (HMW-GS) expression, we isolated four Glu-1Bx promoters from six wheat cultivars exhibiting diverse protein expression levels. The activities of the diverse Glu-1Bx promoters were tested and compared with β-glucuronidase (GUS) reporter fusions. Although all the full-length Glu-1Bx promoters showed endosperm-specific activities, the strongest GUS activity was observed with the 1Bx7OE promoter in both transient expression assays and stable transgenic rice lines. A 43 bp insertion in the 1Bx7OE promoter, which is absent in the 1Bx7 promoter, led to enhanced expression. Analysis of promoter deletion constructs confirmed that a 185 bp MITE (miniature inverted-repeat transposable element) in the 1Bx14 promoter had a weak positive effect on Glu-1Bx expression, and a 54 bp deletion in the 1Bx13 promoter reduced endosperm-specific activity. To investigate the effect of the 43 bp insertion in the 1Bx7OE promoter, a functional marker was developed to screen 505 Chinese varieties and 160 European varieties, and only 1Bx7-type varieties harboring the 43 bp insertion in their promoters showed similar overexpression patterns. Hence, the 1Bx7OE promoter should be important tool in crop genetic engineering as well as in molecular assisted breeding. PMID:25133580

Antibodies against CD20 or B-Cell Receptor Induce Similar Transcription Patterns in Human Lymphoma Cell Lines

PubMed Central

Franke, Andreas; Niederfellner, Gerhard J.; Klein, Christian; Burtscher, Helmut

2011-01-01

Background CD20 is a cell surface protein exclusively expressed on B cells. It is a clinically validated target for Non-Hodgkin's lymphomas (NHL) and autoimmune diseases. The B cell receptor (BCR) plays an important role for development and proliferation of pre-B and B cells. Physical interaction of CD20 with BCR and components of the BCR signaling cascade has been reported but the consequences are not fully understood. Methodology In this study we employed antibodies against CD20 and against the BCR to trigger the respective signaling. These antibodies induced very similar expression patterns of up- and down-regulated genes in NHL cell lines indicating that CD20 may play a role in BCR signaling and vice versa. Two of the genes that were rapidly and transiently induced by both stimuli are CCL3 and CCL4. 4 hours after stimulation the concentration of these chemokines in culture medium reaches a maximum. Spleen tyrosine kinase Syk is a cytoplasmic tyrosine kinase and a key component of BCR signaling. Both siRNA mediated silencing of Syk and inhibition by selective small molecule inhibitors impaired CCL3/CCL4 protein induction after treatment with either anti-CD20 or anti-BCR antibodies. Conclusion Our results suggest that treatment with anti-CD20 antibodies triggers at least partially a BCR activation-like response in NHL cell lines. PMID:21364752

Rapid and transient upregulation of CCL11 (eotaxin-1) in mouse ovary during terminal stages of follicular development.

PubMed

Kuwabara, Yoshimitsu; Katayama, Akira; Igarashi, Tsutomu; Tomiyama, Ryoko; Piao, Hua; Kaneko, Reika; Abe, Takashi; Mine, Katsuya; Akira, Shigeo; Orimo, Hideo; Takeshita, Toshiyuki

2012-05-01

This study aimed to investigate the regulation of expression, localization and physiological role of the CCL11/CCR3 axis in mouse ovary during the periovulatory period. CCL11/CCR3 expression in the mouse ovary after treatment with pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) 48 hr later was assessed in vivo and in 3-dimensional cultures in vitro. Real-time RT-PCR analyses revealed transient CCL11 mRNA upregulation 6 hr after hCG treatment. Immunohistochemical staining of serial ovarian sections demonstrated overlapping expression of CCL11, CCR3 and CD31 endothelial cell marker in the theca-interstitial layer at 10 hr after hCG treatment. In vitro 3-dimensional cultures of periovulatory ovarian tissues demonstrated that treatment with anti-CCL11 neutralizing antibody significantly decreased CD31 transcript. Gonadotropin surge leads to transient CCL11/CCR3 axis upregulation in the ovarian theca-interstitial layer, suggesting that it is involved in periovulatory physiological processes by affecting follicular vessels. © 2012 John Wiley & Sons A/S.

Approximate transient and long time limit solutions for the band broadening induced by the thin sidewall-layer in liquid chromatography columns.

PubMed

Broeckhoven, Ken; Desmet, Gert

2007-11-16

Using a combination of both analytical and numerical techniques, approximate analytical expressions have been established for the transient and long time limit band broadening, originating from the presence of a thin disturbed sidewall layer in liquid chromatography columns, including packed, monolithic as well as microfabricated columns. The established expressions can be used to compare the importance of a thin disturbed sidewall layer with that of other radial heterogeneity effects (such as transcolumn packing density variations due to the relief of packing stresses). The expressions are independent of the actual velocity profile inside the layer as long as the disturbed sidewall layer occupies less than 2.5% of the column width.

Optimatization of transient transformation methods to study gene expression in Musa acuminata (AAA group) cultivar Ambon Lumut

NASA Astrophysics Data System (ADS)

Prayuni, Kinasih; Dwivany, Fenny M.

2015-09-01

Banana is classified as a climateric fruit, whose ripening is regulated by ethylene. Ethylene is synthesized from ACC (1-aminocyclopropane-1-carboxylic acid) by ACC oxidase enzyme which is encoded by ACO gene. Controling an important gene expression in ethylene biosynthesis pathway has became a target to delay the ripening process. Therefore in the previous study we have designed a MaACO-RNAi construct to control MaACO gene expression. In this research, we study the effectiveness of different transient transformation methods to deliver the construct. Direct injection, with or no vaccum infiltration methods were used to deliver MaACO-RNAi construct. All of the methods succesfully deliver the construct into banana fruits based on RT-PCR result.

A Methodology to Determine Self-Similarity, Illustrated by Example: Transient Heat Transfer with Constant Flux

ERIC Educational Resources Information Center

Monroe, Charles; Newman, John

2005-01-01

This simple example demonstrates the physical significance of similarity solutions and the utility of dimensional and asymptotic analysis of partial differential equations. A procedure to determine the existence of similarity solutions is proposed and subsequently applied to transient constant-flux heat transfer. Short-time expressions follow from…

Kainate receptors mediate signaling in both transient and sustained OFF bipolar cell pathways in mouse retina.

PubMed

Borghuis, Bart G; Looger, Loren L; Tomita, Susumu; Demb, Jonathan B

2014-04-30

A fundamental question in sensory neuroscience is how parallel processing is implemented at the level of molecular and circuit mechanisms. In the retina, it has been proposed that distinct OFF cone bipolar cell types generate fast/transient and slow/sustained pathways by the differential expression of AMPA- and kainate-type glutamate receptors, respectively. However, the functional significance of these receptors in the intact circuit during light stimulation remains unclear. Here, we measured glutamate release from mouse bipolar cells by two-photon imaging of a glutamate sensor (iGluSnFR) expressed on postsynaptic amacrine and ganglion cell dendrites. In both transient and sustained OFF layers, cone-driven glutamate release from bipolar cells was blocked by antagonists to kainate receptors but not AMPA receptors. Electrophysiological recordings from bipolar and ganglion cells confirmed the essential role of kainate receptors for signaling in both transient and sustained OFF pathways. Kainate receptors mediated responses to contrast modulation up to 20 Hz. Light-evoked responses in all mouse OFF bipolar pathways depend on kainate, not AMPA, receptors.

Vapor Flow Patterns During a Start-Up Transient in Heat Pipes

NASA Technical Reports Server (NTRS)

Issacci, F.; Ghoniem, N, M.; Catton, I.

1996-01-01

The vapor flow patterns in heat pipes are examined during the start-up transient phase. The vapor core is modelled as a channel flow using a two dimensional compressible flow model. A nonlinear filtering technique is used as a post process to eliminate the non-physical oscillations of the flow variables. For high-input heat flux, multiple shock reflections are observed in the evaporation region. The reflections cause a reverse flow in the evaporation and circulations in the adiabatic region. Furthermore, each shock reflection causes a significant increase in the local pressure and a large pressure drop along the heat pipe.

Weak conservation of structural features in the interfaces of homologous transient protein–protein complexes

PubMed Central

Sudha, Govindarajan; Singh, Prashant; Swapna, Lakshmipuram S; Srinivasan, Narayanaswamy

2015-01-01

Residue types at the interface of protein–protein complexes (PPCs) are known to be reasonably well conserved. However, we show, using a dataset of known 3-D structures of homologous transient PPCs, that the 3-D location of interfacial residues and their interaction patterns are only moderately and poorly conserved, respectively. Another surprising observation is that a residue at the interface that is conserved is not necessarily in the interface in the homolog. Such differences in homologous complexes are manifested by substitution of the residues that are spatially proximal to the conserved residue and structural differences at the interfaces as well as differences in spatial orientations of the interacting proteins. Conservation of interface location and the interaction pattern at the core of the interfaces is higher than at the periphery of the interface patch. Extents of variability of various structural features reported here for homologous transient PPCs are higher than the variation in homologous permanent homomers. Our findings suggest that straightforward extrapolation of interfacial nature and inter-residue interaction patterns from template to target could lead to serious errors in the modeled complex structure. Understanding the evolution of interfaces provides insights to improve comparative modeling of PPC structures. PMID:26311309

Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants.

PubMed

Chen, Yanhui; Han, Yangyang; Zhang, Meng; Zhou, Shan; Kong, Xiangzhu; Wang, Wei

2016-01-01

Expansins are cell wall proteins that are grouped into two main families, α-expansins and β-expansins, and they are implicated in the control of cell extension via the disruption of hydrogen bonds between cellulose and matrix glucans. TaEXPA2 is an α-expansin gene identified in wheat. Based on putative cis-regulatory elements in the TaEXPA2 promoter sequence and the expression pattern induced when polyethylene glycol (PEG) is used to mimic water stress, we hypothesized that TaEXPA2 is involved in plant drought tolerance and plant development. Through transient expression of 35S::TaEXPA2-GFP in onion epidermal cells, TaEXPA2 was localized to the cell wall. Constitutive expression of TaEXPA2 in tobacco improved seed production by increasing capsule number, not seed size, without having any effect on plant growth patterns. The transgenic tobacco exhibited a significantly greater tolerance to water-deficiency stress than did wild-type (WT) plants. We found that under drought stress, the transgenic plants maintained a better water status. The accumulated content of osmotic adjustment substances, such as proline, in TaEXPA2 transgenic plants was greater than that in WT plants. Transgenic plants also displayed greater antioxidative competence as indicated by their lower malondialdehyde (MDA) content, relative electrical conductivity, and reactive oxygen species (ROS) accumulation than did WT plants. This result suggests that the transgenic plants suffer less damage from ROS under drought conditions. The activities of some antioxidant enzymes as well as expression levels of several genes encoding key antioxidant enzymes were higher in the transgenic plants than in the WT plants under drought stress. Collectively, our results suggest that ectopic expression of the wheat expansin gene TaEXPA2 improves seed production and drought tolerance in transgenic tobacco plants.

Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants

PubMed Central

Chen, Yanhui; Han, Yangyang; Zhang, Meng; Zhou, Shan; Kong, Xiangzhu; Wang, Wei

2016-01-01

Expansins are cell wall proteins that are grouped into two main families, α-expansins and β-expansins, and they are implicated in the control of cell extension via the disruption of hydrogen bonds between cellulose and matrix glucans. TaEXPA2 is an α-expansin gene identified in wheat. Based on putative cis-regulatory elements in the TaEXPA2 promoter sequence and the expression pattern induced when polyethylene glycol (PEG) is used to mimic water stress, we hypothesized that TaEXPA2 is involved in plant drought tolerance and plant development. Through transient expression of 35S::TaEXPA2-GFP in onion epidermal cells, TaEXPA2 was localized to the cell wall. Constitutive expression of TaEXPA2 in tobacco improved seed production by increasing capsule number, not seed size, without having any effect on plant growth patterns. The transgenic tobacco exhibited a significantly greater tolerance to water-deficiency stress than did wild-type (WT) plants. We found that under drought stress, the transgenic plants maintained a better water status. The accumulated content of osmotic adjustment substances, such as proline, in TaEXPA2 transgenic plants was greater than that in WT plants. Transgenic plants also displayed greater antioxidative competence as indicated by their lower malondialdehyde (MDA) content, relative electrical conductivity, and reactive oxygen species (ROS) accumulation than did WT plants. This result suggests that the transgenic plants suffer less damage from ROS under drought conditions. The activities of some antioxidant enzymes as well as expression levels of several genes encoding key antioxidant enzymes were higher in the transgenic plants than in the WT plants under drought stress. Collectively, our results suggest that ectopic expression of the wheat expansin gene TaEXPA2 improves seed production and drought tolerance in transgenic tobacco plants. PMID:27073898

Application of dynamic topic models to toxicogenomics data.

PubMed

Lee, Mikyung; Liu, Zhichao; Huang, Ruili; Tong, Weida

2016-10-06

All biological processes are inherently dynamic. Biological systems evolve transiently or sustainably according to sequential time points after perturbation by environment insults, drugs and chemicals. Investigating the temporal behavior of molecular events has been an important subject to understand the underlying mechanisms governing the biological system in response to, such as, drug treatment. The intrinsic complexity of time series data requires appropriate computational algorithms for data interpretation. In this study, we propose, for the first time, the application of dynamic topic models (DTM) for analyzing time-series gene expression data. A large time-series toxicogenomics dataset was studied. It contains over 3144 microarrays of gene expression data corresponding to rat livers treated with 131 compounds (most are drugs) at two doses (control and high dose) in a repeated schedule containing four separate time points (4-, 8-, 15- and 29-day). We analyzed, with DTM, the topics (consisting of a set of genes) and their biological interpretations over these four time points. We identified hidden patterns embedded in this time-series gene expression profiles. From the topic distribution for compound-time condition, a number of drugs were successfully clustered by their shared mode-of-action such as PPARɑ agonists and COX inhibitors. The biological meaning underlying each topic was interpreted using diverse sources of information such as functional analysis of the pathways and therapeutic uses of the drugs. Additionally, we found that sample clusters produced by DTM are much more coherent in terms of functional categories when compared to traditional clustering algorithms. We demonstrated that DTM, a text mining technique, can be a powerful computational approach for clustering time-series gene expression profiles with the probabilistic representation of their dynamic features along sequential time frames. The method offers an alternative way for uncovering hidden patterns embedded in time series gene expression profiles to gain enhanced understanding of dynamic behavior of gene regulation in the biological system.

A zebrafish model for Waardenburg syndrome type IV reveals diverse roles for Sox10 in the otic vesicle.

PubMed

Dutton, Kirsten; Abbas, Leila; Spencer, Joanne; Brannon, Claire; Mowbray, Catriona; Nikaido, Masataka; Kelsh, Robert N; Whitfield, Tanya T

2009-01-01

In humans, mutations in the SOX10 gene are a cause of the auditory-pigmentary disorder Waardenburg syndrome type IV (WS4) and related variants. SOX10 encodes an Sry-related HMG box protein essential for the development of the neural crest; deafness in WS4 and other Waardenburg syndromes is usually attributed to loss of neural-crest-derived melanocytes in the stria vascularis of the cochlea. However, SOX10 is strongly expressed in the developing otic vesicle and so direct roles for SOX10 in the otic epithelium might also be important. Here, we examine the otic phenotype of zebrafish sox10 mutants, a model for WS4. As a cochlea is not present in the fish ear, the severe otic phenotype in these mutants cannot be attributed to effects on this tissue. In zebrafish sox10 mutants, we see abnormalities in all otic placodal derivatives. Gene expression studies indicate deregulated expression of several otic genes, including fgf8, in sox10 mutants. Using a combination of mutant and morphant data, we show that the three sox genes belonging to group E (sox9a, sox9b and sox10) provide a link between otic induction pathways and subsequent otic patterning: they act redundantly to maintain sox10 expression throughout otic tissue and to restrict fgf8 expression to anterior macula regions. Single-cell labelling experiments indicate a small and transient neural crest contribution to the zebrafish ear during normal development, but this is unlikely to account for the strong defects seen in the sox10 mutant. We discuss the implication that the deafness in WS4 patients with SOX10 mutations might reflect a haploinsufficiency for SOX10 in the otic epithelium, resulting in patterning and functional abnormalities in the inner ear.

Effects of hypoxic preconditioning on expression of transcription factor NGFI-A in the rat brain after unavoidable stress in the "learned helplessness" model.

PubMed

Baranova, K A; Rybnikova, E A; Mironova, V I; Samoilov, M O

2010-07-01

We report here our immunocytochemical studies establishing that the development of a depression-like state in rats following unavoidable stress in a "learned helplessness" model is accompanied by stable activation of the expression of transcription factor NGFI-A in the dorsal hippocampus (field CA1) and the magnocellular paraventricular nucleus of the hypothalamus, along with an early wave of post-stress expression, which died down rapidly, in the ventral hippocampus (the dentate gyrus) and a long period of up to five days of high-level expression in the neocortex. In rats subjected to three sessions of preconditioning consisting of moderate hypobaric hypoxia (360 mmHg, 2 h, with intervals of 24 h), which did not form depression in these circumstances, there were significant changes in the dynamics of immunoreactive protein content in the hippocampus, with a stable increase in expression in the ventral hippocampus and only transient and delayed (by five days) expression in field CA1. In the neocortex (layer II), preconditioning eliminated the effects of stress, preventing prolongation of the first wave of NGFI-A expression to five days, while in the magnocellular hypothalamus, conversely, preconditioning stimulated a second (delayed) wave of the expression of this transcription factor. The pattern of NGFI-A expression in the hippocampus, neocortex, and hypothalamus seen in non-preconditioned rats appears to reflect the pathological reaction to aversive stress, which, rather than adaptation, produced depressive disorders. Post-stress modification of the expression of the product of the early gene NGFI-A in the brain induced by hypoxic preconditioning probably plays an important role in increased tolerance to severe psychoemotional stresses and is an important component of antidepressant mechanisms.

miR-122 negatively correlates with liver fibrosis as detected by histology and FibroScan

PubMed Central

Halász, Tünde; Horváth, Gábor; Pár, Gabriella; Werling, Klára; Kiss, András; Schaff, Zsuzsa; Lendvai, Gábor

2015-01-01

AIM: To investigate whether expression of selected miRNAs obtained from fibrotic liver biopsies correlate with fibrosis stage. METHODS: Altogether, 52 patients were enrolled in the study representing various etiologic backgrounds of fibrosis: 24 cases with chronic hepatitis infections (types B, C), 19 with autoimmune liver diseases (autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, overlapping syndrome cases), and 9 of mixed etiology (alcoholic and nonalcoholic steatosis, cryptogenic cases). Severity of fibrosis was determined by both histologic staging using the METAVIR scoring system and noninvasive transient elastography. Following RNA isolation, expression levels of miR-21, miR-122, miR-214, miR-221, miR-222, and miR-224 were determined using TaqMan MicroRNA Assays applying miR-140 as the reference. Selection of miRNAs was based on their characteristic up- or downregulation observed in hepatocellular carcinoma. Relative expression of miRNAs was correlated with fibrosis stage and liver stiffness (LS) value measured by transient elastography, as well as with serum alanine aminotransferase (ALT) level. RESULTS: The expression of individual miRNAs showed deregulated patterns in stages F1-F4 as compared with stage F0, but only the reduced level of miR-122 in stage F4 was statistically significant (P < 0.04). When analyzing miRNA expression in relation to fibrosis, levels of miR-122 and miR-221 showed negative correlations with fibrosis stage, and miR-122 was found to correlate negatively and miR-224 positively with LS values (all P < 0.05). ALT levels displayed a positive correlation with miR-21 (P < 0.04). Negative correlations were observed in the fibrosis samples of mixed etiology between miR-122 and fibrosis stage and LS values (P < 0.05), and in the samples of chronic viral hepatitis, between miR-221 and fibrosis stage (P < 0.01), whereas miR-21 showed positive correlation with ALT values in the samples of autoimmune liver diseases (P < 0.03). The results also revealed a strong correlation between fibrosis stage and LS values (P < 0.01) when etiology of fibrosis was not taken into account. CONCLUSION: Reduced expression of miR-122 in advanced fibrosis and its correlation with fibrosis stage and LS values seem to be characteristic of hepatic fibrosis of various etiologies. PMID:26167081

miR-122 negatively correlates with liver fibrosis as detected by histology and FibroScan.

PubMed

Halász, Tünde; Horváth, Gábor; Pár, Gabriella; Werling, Klára; Kiss, András; Schaff, Zsuzsa; Lendvai, Gábor

2015-07-07

To investigate whether expression of selected miRNAs obtained from fibrotic liver biopsies correlate with fibrosis stage. Altogether, 52 patients were enrolled in the study representing various etiologic backgrounds of fibrosis: 24 cases with chronic hepatitis infections (types B, C), 19 with autoimmune liver diseases (autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, overlapping syndrome cases), and 9 of mixed etiology (alcoholic and nonalcoholic steatosis, cryptogenic cases). Severity of fibrosis was determined by both histologic staging using the METAVIR scoring system and noninvasive transient elastography. Following RNA isolation, expression levels of miR-21, miR-122, miR-214, miR-221, miR-222, and miR-224 were determined using TaqMan MicroRNA Assays applying miR-140 as the reference. Selection of miRNAs was based on their characteristic up- or downregulation observed in hepatocellular carcinoma. Relative expression of miRNAs was correlated with fibrosis stage and liver stiffness (LS) value measured by transient elastography, as well as with serum alanine aminotransferase (ALT) level. The expression of individual miRNAs showed deregulated patterns in stages F1-F4 as compared with stage F0, but only the reduced level of miR-122 in stage F4 was statistically significant (P < 0.04). When analyzing miRNA expression in relation to fibrosis, levels of miR-122 and miR-221 showed negative correlations with fibrosis stage, and miR-122 was found to correlate negatively and miR-224 positively with LS values (all P < 0.05). ALT levels displayed a positive correlation with miR-21 (P < 0.04). Negative correlations were observed in the fibrosis samples of mixed etiology between miR-122 and fibrosis stage and LS values (P < 0.05), and in the samples of chronic viral hepatitis, between miR-221 and fibrosis stage (P < 0.01), whereas miR-21 showed positive correlation with ALT values in the samples of autoimmune liver diseases (P < 0.03). The results also revealed a strong correlation between fibrosis stage and LS values (P < 0.01) when etiology of fibrosis was not taken into account. Reduced expression of miR-122 in advanced fibrosis and its correlation with fibrosis stage and LS values seem to be characteristic of hepatic fibrosis of various etiologies.

Regulated viral BDNF delivery in combination with Schwann cells promotes axonal regeneration through capillary alginate hydrogels after spinal cord injury.

PubMed

Liu, Shengwen; Sandner, Beatrice; Schackel, Thomas; Nicholson, LaShae; Chtarto, Abdelwahed; Tenenbaum, Liliane; Puttagunta, Radhika; Müller, Rainer; Weidner, Norbert; Blesch, Armin

2017-09-15

Grafting of cell-seeded alginate capillary hydrogels into a spinal cord lesion site provides an axonal bridge while physically directing regenerating axonal growth in a linear pattern. However, without an additional growth stimulus, bridging axons fail to extend into the distal host spinal cord. Here we examined whether a combinatory strategy would support regeneration of descending axons across a cervical (C5) lateral hemisection lesion in the rat spinal cord. Following spinal cord transections, Schwann cell (SC)-seeded alginate hydrogels were grafted to the lesion site and AAV5 expressing brain-derived neurotrophic factor (BDNF) under control of a tetracycline-regulated promoter was injected caudally. In addition, we examined whether SC injection into the caudal spinal parenchyma would further enhance regeneration of descending axons to re-enter the host spinal cord. Our data show that both serotonergic and descending axons traced by biotinylated dextran amine (BDA) extend throughout the scaffolds. The number of regenerating axons is significantly increased when caudal BDNF expression is activated and transient BDNF delivery is able to sustain axons after gene expression is switched off. Descending axons are confined to the caudal graft/host interface even with continuous BDNF expression for 8weeks. Only with a caudal injection of SCs, a pathway facilitating axonal regeneration through the host/graft interface is generated allowing axons to successfully re-enter the caudal spinal cord. Recovery from spinal cord injury is poor due to the limited regeneration observed in the adult mammalian central nervous system. Biomaterials, cell transplantation and growth factors that can guide axons across a lesion site, provide a cellular substrate, stimulate axon growth and have shown some promise in increasing the growth distance of regenerating axons. In the present study, we combined an alginate biomaterial with linear channels with transplantation of Schwann cells within and beyond the lesion site and injection of a regulatable vector for the transient expression of brain-derived neurotrophic factor (BDNF). Our data show that only with the full combination axons extend across the lesion site and that expression of BDNF beyond 4weeks does not further increase the number of regenerating axons. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Peripheral inflammation induces up-regulation of TRPV2 expression in rat DRG.

PubMed

Shimosato, Goshun; Amaya, Fumimasa; Ueda, Masashi; Tanaka, Yoshifumi; Decosterd, Isabelle; Tanaka, Masaki

2005-12-15

The transient receptor potential vanilloid subfamily member 2 (TRPV2) is a cation channel activated by temperatures above 52 degrees C. To analyze the contribution of TRPV2 to the development of inflammation-induced hyperalgesia, the expression of TRPV2 in primary sensory neurons was analyzed after intraplantar injection of complete Freund's adjuvant (CFA). Using specific antibodies, an increase in TRPV2-expressing neurons was identified after inflammation. TRPV2 expression is concentrated in a subset of medium-sized dorsal root ganglion neurons, independent of transient receptor potential vanilloid subfamily member 1 (TRPV1) expression. A similar distribution of TRPV2 was observed after inflammation. Intraplantar injection of nerve growth factor increased TRPV1 expression but not TRPV2, suggesting that induction of TRPV2 expression is driven by a mechanism distinct from that for TRPV1. Heat hyperalgesia assessment after chemical desensitization of TRPV1 by resiniferatoxin demonstrates a possible role for TRPV2 in inflammation at high temperatures (>56 degrees C). These results suggest that TRPV2 upregulation contributes to peripheral sensitization during inflammation and is responsible for pain hypersensitivity to noxious high temperature stimuli.

Novel Analysis Software for Detecting and Classifying Ca2+ Transient Abnormalities in Stem Cell-Derived Cardiomyocytes

PubMed Central

Penttinen, Kirsi; Siirtola, Harri; Àvalos-Salguero, Jorge; Vainio, Tiina; Juhola, Martti; Aalto-Setälä, Katriina

2015-01-01

Comprehensive functioning of Ca2+ cycling is crucial for excitation–contraction coupling of cardiomyocytes (CMs). Abnormal Ca2+ cycling is linked to arrhythmogenesis, which is associated with cardiac disorders and heart failure. Accordingly, we have generated spontaneously beating CMs from induced pluripotent stem cells (iPSC) derived from patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), which is an inherited and severe cardiac disease. Ca2+ cycling studies have revealed substantial abnormalities in these CMs. Ca2+ transient analysis performed manually lacks accepted analysis criteria, and has both low throughput and high variability. To overcome these issues, we have developed a software tool, AnomalyExplorer based on interactive visualization, to assist in the classification of Ca2+ transient patterns detected in CMs. Here, we demonstrate the usability and capability of the software, and we also compare the analysis efficiency to manual analysis. We show that AnomalyExplorer is suitable for detecting normal and abnormal Ca2+ transients; furthermore, this method provides more defined and consistent information regarding the Ca2+ abnormality patterns and cell line specific differences when compared to manual analysis. This tool will facilitate and speed up the analysis of CM Ca2+ transients, making it both more accurate and user-independent. AnomalyExplorer can be exploited in Ca2+ cycling analysis to study basic disease pathology and the effects of different drugs. PMID:26308621

Novel Analysis Software for Detecting and Classifying Ca2+ Transient Abnormalities in Stem Cell-Derived Cardiomyocytes.

PubMed

Penttinen, Kirsi; Siirtola, Harri; Àvalos-Salguero, Jorge; Vainio, Tiina; Juhola, Martti; Aalto-Setälä, Katriina

2015-01-01

Comprehensive functioning of Ca2+ cycling is crucial for excitation-contraction coupling of cardiomyocytes (CMs). Abnormal Ca2+ cycling is linked to arrhythmogenesis, which is associated with cardiac disorders and heart failure. Accordingly, we have generated spontaneously beating CMs from induced pluripotent stem cells (iPSC) derived from patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), which is an inherited and severe cardiac disease. Ca2+ cycling studies have revealed substantial abnormalities in these CMs. Ca2+ transient analysis performed manually lacks accepted analysis criteria, and has both low throughput and high variability. To overcome these issues, we have developed a software tool, AnomalyExplorer based on interactive visualization, to assist in the classification of Ca2+ transient patterns detected in CMs. Here, we demonstrate the usability and capability of the software, and we also compare the analysis efficiency to manual analysis. We show that AnomalyExplorer is suitable for detecting normal and abnormal Ca2+ transients; furthermore, this method provides more defined and consistent information regarding the Ca2+ abnormality patterns and cell line specific differences when compared to manual analysis. This tool will facilitate and speed up the analysis of CM Ca2+ transients, making it both more accurate and user-independent. AnomalyExplorer can be exploited in Ca2+ cycling analysis to study basic disease pathology and the effects of different drugs.

Transient rheology of the uppermost mantle beneath the Mojave Desert, California

USGS Publications Warehouse

Pollitz, F.F.

2003-01-01

Geodetic data indicate that the M7.1 Hector Mine, California, earthquake was followed by a brief period (a few weeks) of rapid deformation preceding a prolonged phase of slower deformation. We find that the signal contained in continuous and campaign global positioning system data for 2.5 years after the earthquake may be explained with a transient rheology. Quantitative modeling of these data with allowance for transient (linear biviscous) rheology in the lower crust and upper mantle demonstrates that transient rheology in the upper mantle is dominant, its material properties being typified by two characteristic relaxation times ???0.07 and ???2 years. The inferred mantle rheology is a Jeffreys solid in which the transient and steady-state shear moduli are equal. Consideration of a simpler viscoelastic model with a linear univiscous rheology (2 fewer parameters than a biviscous model) shows that it consistently underpredicts the amplitude of the first ???3 months signal, and allowance for a biviscous rheology is significant at the 99.0% confidence level. Another alternative model - deep postseismic afterslip beneath the coseismic rupture - predicts a vertical velocity pattern opposite to the observed pattern at all time periods considered. Despite its plausibility, the advocated biviscous rheology model is non-unique and should be regarded as a viable alternative to the non-linear mantle rheology model for governing postseismic flow beneath the Mojave Desert. Published by Elsevier B.V.

Differing patterns of neurotrophin-receptor expressing neurons allow distinction of the transient Frorieps' ganglia from normal DRG before morphological differences appear.

PubMed

Avivi, Camila; Goldstein, Ronald S

2003-10-10

The Frorieps' ganglia are dorsal root ganglia (DRG) that form and then degenerate during normal embryonic development of amniotes. Their degeneration or survival has been shown to be modulated by modifying expression of Hox-family and other genes involved in pattern formation, and by the mesodermal microenvironment of the cranial somites in which they develop. In ovo application of the neurotrophin NGF partially rescues DRG2 from degeneration. To further examine the potential role of neurotrophins in the life cycle of Frorieps' DRG we have now quantified the numbers of neurons expressing neurotrophin receptors trkA and trkC in avian Frorieps' ganglia (DRG2) and normal cervical DRG (DRG5). We have found that the Frorieps' DRG are different from normal DRG in terms of the numbers of neurons expressing these receptors. trkC-expressing neurons are generally lacking in DRG2, this is the earliest (St 18, E2.5) described difference between DRG2 and normal DRG, preceding morphological differences between these ganglia that appear at St 20. The difference between DRG2 and DRG5 in terms of numbers of trkA-expressing neurons is evident only at later embryonic stages, where DRG2 contains a higher proportion of trkA neurons than normal cervical DRG. The few trkC+ neurons present late in DRG2 development are not concentrated in the VL portion of the ganglion, the zone where trkC+ neurons are generally found in normal DRG. We also find that DRG2 neurons are smaller than those of normal DRG, this is true for both trkA+ and trkC+ populations. These data together therefore suggest that the neurons that survive in the Frorieps' ganglia at later stages belong almost exclusively to the trkA-expressing DM class DRG neurons. We further find that the differences in the populations of trkA/trkC between DRG2 and DRG5 result from signals from the mesodermal microenvironment, since DRG arising in cranial somites transplanted caudally contain few trkC+ neurons and a higher proportion of trkA+ cells than contralateral controls.

The TIR domain of TIR-NB-LRR resistance proteins is a signaling domain involved in cell death induction.

PubMed

Swiderski, Michal R; Birker, Doris; Jones, Jonathan D G

2009-02-01

In plants, the TIR (toll interleukin 1 receptor) domain is found almost exclusively in nucleotide-binding (NB) leucine-rich repeat resistance proteins and their truncated homologs, and has been proposed to play a signaling role during resistance responses mediated by TIR containing R proteins. Transient expression in Nicotiana benthamiana leaves of "TIR + 80", the RPS4 truncation without the NB-ARC domain, leads to EDS1-, SGT1-, and HSP90-dependent cell death. Transgenic Arabidopsis plants expressing the RPS4 TIR+80 from either dexamethasone or estradiol-inducible promoters display inducer-dependent cell death. Cell death is also elicited by transient expression of similarly truncated constructs from two other R proteins, RPP1A and At4g19530, but is not elicited by similar constructs representing RPP2A and RPP2B proteins. Site-directed mutagenesis of the RPS4 TIR domain identified many loss-of-function mutations but also revealed several gain-of function substitutions. Lack of cell death induction by the E160A substitution suggests that amino acids outside of the TIR domain contribute to cell death signaling in addition to the TIR domain itself. This is consistent with previous observations that the TIR domain itself is insufficient to induce cell death upon transient expression.

Complex pattern of interaction between in utero hypoxia-ischemia and intra-amniotic inflammation disrupts brain development and motor function

PubMed Central

2014-01-01

Background Infants born preterm commonly suffer from a combination of hypoxia-ischemia (HI) and infectious perinatal inflammatory insults that lead to cerebral palsy, cognitive delay, behavioral issues and epilepsy. Using a novel rat model of combined late gestation HI and lipopolysaccharide (LPS)-induced inflammation, we tested our hypothesis that inflammation from HI and LPS differentially affects gliosis, white matter development and motor impairment during the first postnatal month. Methods Pregnant rats underwent laparotomy on embryonic day 18 and transient systemic HI (TSHI) and/or intra-amniotic LPS injection. Shams received laparotomy and anesthesia only. Pups were born at term. Immunohistochemistry with stereological estimates was performed to assess regional glial loads, and western blots were performed for protein expression. Erythropoietin ligand and receptor levels were quantified using quantitative PCR. Digigait analysis detected gait deficits. Statistical analysis was performed with one-way analysis of variance and post-hoc Bonferonni correction. Results Microglial and astroglial immunolabeling are elevated in TSHI + LPS fimbria at postnatal day 2 compared to sham (both P < 0.03). At postnatal day 15, myelin basic protein expression is reduced by 31% in TSHI + LPS pups compared to shams (P < 0.05). By postnatal day 28, white matter injury shifts from the acute injury pattern to a chronic injury pattern in TSHI pups only. Both myelin basic protein expression (P < 0.01) and the phosphoneurofilament/neurofilament ratio, a marker of axonal dysfunction, are reduced in postnatal day 28 TSHI pups (P < 0.001). Erythropoietin ligand to receptor ratios differ between brains exposed to TSHI and LPS. Gait analyses reveal that all groups (TSHI, LPS and TSHI + LPS) are ataxic with deficits in stride, paw placement, gait consistency and coordination (all P < 0.001). Conclusions Prenatal TSHI and TSHI + LPS lead to different patterns of injury with respect to myelination, axon integrity and gait deficits. Dual injury leads to acute alterations in glial response and cellular inflammation, while TSHI alone causes more prominent chronic white matter and axonal injury. Both injuries cause significant gait deficits. Further study will contribute to stratification of injury mechanisms in preterm infants, and guide the use of promising therapeutic interventions. PMID:25082427

Calcium Dynamics in Basal Dendrites of Layer 5A and 5B Pyramidal Neurons Is Tuned to the Cell-Type Specific Physiological Action Potential Discharge

PubMed Central

Krieger, Patrik; de Kock, Christiaan P. J.; Frick, Andreas

2017-01-01

Layer 5 (L5) is a major neocortical output layer containing L5A slender-tufted (L5A-st) and L5B thick-tufted (L5B-tt) pyramidal neurons. These neuron types differ in their in vivo firing patterns, connectivity and dendritic morphology amongst other features, reflecting their specific functional role within the neocortical circuits. Here, we asked whether the active properties of the basal dendrites that receive the great majority of synaptic inputs within L5 differ between these two pyramidal neuron classes. To quantify their active properties, we measured the efficacy with which action potential (AP) firing patterns backpropagate along the basal dendrites by measuring the accompanying calcium transients using two-photon laser scanning microscopy in rat somatosensory cortex slices. For these measurements we used both “artificial” three-AP patterns and more complex physiological AP patterns that were previously recorded in anesthetized rats in L5A-st and L5B-tt neurons in response to whisker stimulation. We show that AP patterns with relatively few APs (3APs) evoke a calcium response in L5B-tt, but not L5A-st, that is dependent on the temporal pattern of the three APs. With more complex in vivo recorded AP patterns, the average calcium response was similar in the proximal dendrites but with a decay along dendrites (measured up to 100 μm) of L5B-tt but not L5A-st neurons. Interestingly however, the whisker evoked AP patterns—although very different for the two cell types—evoke similar calcium responses. In conclusion, although the effectiveness with which different AP patterns evoke calcium transients vary between L5A-st and L5B-tt cell, the calcium influx appears to be tuned such that whisker-evoked calcium transients are within the same dynamic range for both cell types. PMID:28744201

Urban Infrastructure, Channel-Floodplain Morphology and Flood Flow Patterns

NASA Astrophysics Data System (ADS)

Miller, A. J.; Smith, J. A.; Nelson, C. B.

2006-12-01

The relationship between the channel and the floodplain in urban settings is heavily influenced by (1) altered watershed hydrologic response and frequency distribution of flows, (2) channel enlargement resulting from altered hydrology under conditions of limited sediment supply, (3) direct modification of channels and floodplains for purposes of erosion mitigation, flood protection, commercial development and creation of public amenities, (4) valley constrictions and flow obstructions associated with bridges, culverts, road embankments and other types of floodplain encroachment causing fragmentation or longitudinal segmentation of the riparian corridor. Field observation of inundation patterns associated with recurring floods in the Baltimore metropolitan area is used in combination with 2-dimensional hydraulic modeling to simulate patterns of floodplain inundation and to explore the relationships between magnitude and shape of the flood hydrograph, morphology of the urban channel-floodplain system, and the frequency and extent of floodplain inundation. Case studies include a July 2004 flood associated with a 300-year 2-hour rainfall in a small (14.2 km2) urban watershed, as well as several other events caused by summer thunderstorms with shorter recurrence intervals that generated an extraordinary flood response. The influence of urban infrastructure on flood inundation and flow patterns is expressed in terms of altered (and hysteretic) stage-discharge relationships, stepped flood profiles, rapid longitudinal attenuation of flood waves, and transient flow reversals at confluences and constrictions. Given the current level of interest in restoration measures these patterns merit consideration in planning future development and mitigation efforts.

Analysis of gene expression during parabolic flights reveals distinct early gravity responses in Arabidopsis roots.

PubMed

Aubry-Hivet, D; Nziengui, H; Rapp, K; Oliveira, O; Paponov, I A; Li, Y; Hauslage, J; Vagt, N; Braun, M; Ditengou, F A; Dovzhenko, A; Palme, K

2014-01-01

Plant roots are among most intensively studied biological systems in gravity research. Altered gravity induces asymmetric cell growth leading to root bending. Differential distribution of the phytohormone auxin underlies root responses to gravity, being coordinated by auxin efflux transporters from the PIN family. The objective of this study was to compare early transcriptomic changes in roots of Arabidopsis thaliana wild type, and pin2 and pin3 mutants under parabolic flight conditions and to correlate these changes to auxin distribution. Parabolic flights allow comparison of transient 1-g, hypergravity and microgravity effects in living organisms in parallel. We found common and mutation-related genes differentially expressed in response to transient microgravity phases. Gene ontology analysis of common genes revealed lipid metabolism, response to stress factors and light categories as primarily involved in response to transient microgravity phases, suggesting that fundamental reorganisation of metabolic pathways functions upstream of a further signal mediating hormonal network. Gene expression changes in roots lacking the columella-located PIN3 were stronger than in those deprived of the epidermis and cortex cell-specific PIN2. Moreover, repetitive exposure to microgravity/hypergravity and gravity/hypergravity flight phases induced an up-regulation of auxin responsive genes in wild type and pin2 roots, but not in pin3 roots, suggesting a critical function of PIN3 in mediating auxin fluxes in response to transient microgravity phases. Our study provides important insights towards understanding signal transduction processes in transient microgravity conditions by combining for the first time the parabolic flight platform with the transcriptome analysis of different genetic mutants in the model plant, Arabidopsis. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

Redox signaling in remote ischemic preconditioning-induced cardioprotection: Evidences and mechanisms.

PubMed

Singh, Lovedeep; Randhawa, Puneet Kaur; Singh, Nirmal; Jaggi, Amteshwar Singh

2017-08-15

Reactive oxygen species are the reactive molecules that are derived from molecular oxygen and play an important role as redox signaling molecules to confer cardioprotection. Various scientists have demonstrated the key role of redox signaling in cardioprotection by showing a transient increase in their levels during remote ischemic preconditioning (RIPC) phase. The transient increase in reactive oxygen species levels during remote preconditioning phase may take place either through activation of K ATP channels or through increased nitric oxide (NO) production. A transient increase in reactive oxygen species during preconditioning may also increase the expression of heat shock proteins (HSP), the level of antioxidant enzymes and decrease the expression of inflammatory genes (Egr-1) during ischemia-reperfusion phase to confer cardioprotection. The present review describes the role of redox signaling in RIPC-induced cardioprotective effect with possible mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo

PubMed Central

Lewis, Jo E.; Brameld, John M.; Hill, Phil; Barrett, Perry; Ebling, Francis J.P.; Jethwa, Preeti H.

2015-01-01

Introduction The viral 2A sequence has become an attractive alternative to the traditional internal ribosomal entry site (IRES) for simultaneous over-expression of two genes and in combination with recombinant adeno-associated viruses (rAAV) has been used to manipulate gene expression in vitro. New method To develop a rAAV construct in combination with the viral 2A sequence to allow long-term over-expression of the vgf gene and fluorescent marker gene for tracking of the transfected neurones in vivo. Results Transient transfection of the AAV plasmid containing the vgf gene, viral 2A sequence and eGFP into SH-SY5Y cells resulted in eGFP fluorescence comparable to a commercially available reporter construct. This increase in fluorescent cells was accompanied by an increase in VGF mRNA expression. Infusion of the rAAV vector containing the vgf gene, viral 2A sequence and eGFP resulted in eGFP fluorescence in the hypothalamus of both mice and Siberian hamsters, 32 weeks post infusion. In situ hybridisation confirmed that the location of VGF mRNA expression in the hypothalamus corresponded to the eGFP pattern of fluorescence. Comparison with old method The viral 2A sequence is much smaller than the traditional IRES and therefore allowed over-expression of the vgf gene with fluorescent tracking without compromising viral capacity. Conclusion The use of the viral 2A sequence in the AAV plasmid allowed the simultaneous expression of both genes in vitro. When used in combination with rAAV it resulted in long-term over-expression of both genes at equivalent locations in the hypothalamus of both Siberian hamsters and mice, without any adverse effects. PMID:26300182

Short-term hyperglycaemia causes non-reversible changes in arterial gene expression in a fully 'switchable' in vivo mouse model of diabetes.

PubMed

Zervou, S; Wang, Y-F; Laiho, A; Gyenesei, A; Kytömäki, L; Hermann, R; Abouna, S; Epstein, D; Pelengaris, S; Khan, M

2010-12-01

Irreversible arterial damage due to early effects of hypo- or hyperglycaemia could account for the limited success of glucose-lowering treatments in preventing cardiovascular disease (CVD) events. We hypothesised that even brief hypo- or hyperglycaemia could adversely affect arterial gene expression and that these changes, moreover, might not be fully reversible. By controlled activation of a 'switchable' c-Myc transgene in beta cells, adult pIns-c-MycER(TAM) mice were rendered transiently hypo- and then hyperglycaemic, after which they were allowed to recover for up to 3 months. Immediate and sequential changes in aortic global gene expression from normal glycaemia through hypo- and hyperglycaemia to recovery were assessed. Gene expression was compared with that of normoglycaemic transgenic and tamoxifen-treated wild-type controls. Overall, expression of 95 genes was significantly affected by moderate hypoglycaemia (glucose down to 2.5 mmol/l), whereas over 769 genes were affected by hyperglycaemia. Genes and pathways activated included several involved in atherogenic processes, such as inflammation and arterial calcification. Although expression of many genes recovered to initial pre-exposure levels when hyperglycaemia was corrected (74.9%), in one in four genes this did not occur. Quantitative reverse transcriptase PCR and immunohistochemistry verified the gene expression patterns of key molecules, as shown by global gene arrays. Short-term exposure to hyperglycaemia can cause deleterious and persistent changes in arterial gene expression in vivo. Brief hypoglycaemia also adversely affects gene expression, although less substantially. Together, these results suggest that early correction of hyperglycaemia and avoidance of hypoglycaemia may both be necessary to avoid excess CVD risk in diabetes.

Tombusvirus-based vector systems to permit over-expression of genes or that serve as sensors of antiviral RNA silencing in plants.

PubMed

Shamekova, Malika; Mendoza, Maria R; Hsieh, Yi-Cheng; Lindbo, John; Omarov, Rustem T; Scholthof, Herman B

2014-03-01

A next generation Tomato bushy stunt virus (TBSV) coat protein gene replacement vector system is described that can be applied by either RNA inoculation or through agroinfiltration. A vector expressing GFP rapidly yields high levels of transient gene expression in inoculated leaves of various plant species, as illustrated for Nicotiana benthamiana, cowpea, tomato, pepper, and lettuce. A start-codon mutation to down-regulate the dose of the P19 silencing suppressor reduces GFP accumulation, whereas mutations that result in undetectable levels of P19 trigger rapid silencing of GFP. Compared to existing virus vectors the TBSV system has a unique combination of a very broad host range, rapid and high levels of replication and gene expression, and the ability to regulate its suppressor. These features are attractive for quick transient assays in numerous plant species for over-expression of genes of interest, or as a sensor to monitor the efficacy of antiviral RNA silencing. Copyright © 2014. Published by Elsevier Inc.

Production of human rotavirus and Salmonella antigens in plants and elicitation of fljB-specific humoral responses in mice.

PubMed

Bergeron-Sandoval, Louis-Philippe; Girard, Aurélie; Ouellet, François; Archambault, Denis; Sarhan, Fathey

2011-02-01

A Nicotiana benthamiana transient expression system was used to express single antigen and dimeric combinations of the human rotavirus (HRV) VP7 and a truncated VP4 (VP4Δ) proteins fused with Salmonella typhimurium's flagellin fljB subunit. Immunoblot analyses using rabbit antibodies generated against these proteins demonstrated that the constructs were successfully expressed with yields ranging from 0.85 to 31.97 μg of recombinant protein per gram of fresh leaf tissue. Expressing the single and dimeric antigens has no effect on plant growth and development except for VP7 and VP4Δ::VP7, which show mild necrotic lesions. Immunization of mice with proteins from leaves transformed with constructs bearing the fljB moiety elicited an fljB-specific humoral response. The Nicotiana benthamiana transient system is efficient to express multiple combinations of pathogen proteins and demonstrates the potential of generating a Salmonella typhimurium subunit vaccine in plants.

Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses

PubMed Central

Raikwar, Shailendra; Srivastava, Vineet K.; Gill, Sarvajeet S.; Tuteja, Renu; Tuteja, Narendra

2015-01-01

Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress conditions. Here, we report the in silico analysis of novel stress inducible promoter of Oryza sativa XPB2 (OsXPB2). The in vivo validation of functionality/activity of OsXPB2 promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. The present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration, or cold) and hormone (Auxin, ABA, or MeJA) induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA, or ABA responsive, respectively. Functional analysis was done by Agrobacterium-mediated transient assay using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present findings suggest that OsXPB2 promoter is a multi-stress inducible promoter and has potential applications in sustainable crop production under abiotic stresses by regulating desirable pattern of gene expression. PMID:26734018

Fermented soybeans, Chungkookjang, prevent hippocampal cell death and β-cell apoptosis by decreasing pro-inflammatory cytokines in gerbils with transient artery occlusion

PubMed Central

Park, Sunmin; Kim, Da Sol; Moon, Bo Reum

2015-01-01

Since Chungkookjang, a short-term fermented soybean, is known to improve glucose metabolism and antioxidant activity, it may prevent the neurological symptoms and glucose disturbance induced by artery occlusion. We investigated the protective effects and mechanisms of traditional (TFC) and standardized Chungkookjang fermented with Bacillus licheniformis (BLFC) against ischemia/reperfusion damage in the hippocampal CA1 region and against hyperglycemia after transient cerebral ischemia in gerbils. Gerbils were subjected to either an occlusion of the bilateral common carotid arteries for 8 min to render them ischemic or a sham operation. Ischemic gerbils were fed either a 40% fat diet containing 10% of either cooked soybean (CSB), TFC, or BLFC for 28 days. Neuronal cell death and cytokine expression in the hippocampus, neurological deficit, serum cytokine levels, and glucose metabolism were measured. TFC and BLFC contained more isoflavonoid aglycones than CSB. Artery occlusion increased the expressions of IL-1β and TNF-α as well as cell death in the hippocampal CA1 region and induced severe neurological symptoms. CSB, TFC, and BLFC prevented the neuronal cell death and the symptoms such as dropped eyelid, bristling hair, reduced muscle tone and flexor reflex, and abnormal posture and walking patterns, and suppressed cytokine expressions. CSB was less effective than TFC and BLFC. Artery occlusion induced glucose intolerance due to decreased insulin secretion and β-cell mass. TFC and BLFC prevented the impairment of glucose metabolism by artery occlusion. Especially TFC and BLFC increased β-cell proliferation and suppressed the β-cell apoptosis by suppressing TNF-α and IL-1β which in turn decreased cleaved caspase-3 that caused apoptosis. In conclusion, TFC and BLFC may prevent and alleviate neuronal cell death in the hippocampal CA1 region and neurological symptoms and poststroke hyperglycemia in gerbils with artery occlusion. This might be associated with increased isoflavonoid aglycones. PMID:26468168

Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses.

PubMed

Raikwar, Shailendra; Srivastava, Vineet K; Gill, Sarvajeet S; Tuteja, Renu; Tuteja, Narendra

2015-01-01

Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress conditions. Here, we report the in silico analysis of novel stress inducible promoter of Oryza sativa XPB2 (OsXPB2). The in vivo validation of functionality/activity of OsXPB2 promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. The present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration, or cold) and hormone (Auxin, ABA, or MeJA) induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA, or ABA responsive, respectively. Functional analysis was done by Agrobacterium-mediated transient assay using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present findings suggest that OsXPB2 promoter is a multi-stress inducible promoter and has potential applications in sustainable crop production under abiotic stresses by regulating desirable pattern of gene expression.

Proteomic analysis reveals protein expression differences in Escherichia coli strains associated with persistent versus transient mastitis

USDA-ARS?s Scientific Manuscript database

Escherichia coli is a leading cause of bacterial mastitis in dairy cattle. Typically this infection is transient in nature, causing an infection that lasts 2-3 days. However, in a minority of cases, E. coli has been shown to cause a persistent intramammary infection. The mechanisms that allow for...

Sumoylation Dynamics During Keratinocyte Differentiation

PubMed Central

Deyrieux, Adeline F.; Rosas-Acosta, Germán; Ozbun, Michelle A.; Wilson, Van G.

2012-01-01

Summary SUMO modification regulates the activity of numerous transcription factors that have a direct role in cell cycle progression, apoptosis, cellular proliferation, and development, but its role in differentiation processes is less clear. Keratinocyte differentiation requires the coordinated activation of a series of transcription factors, and as several critical keratinocyte transcription factors are known to be SUMO substrates, we investigated the role of sumoylation in keratinocyte differentiation. In a human keratinocyte cell line model (HaCaT cells), calcium-induced differentiation led to the transient and coordinated transcriptional activation of the genes encoding critical sumoylation system components, including SAE1, SAE2, Ubc9, SENP1, Miz-1 (PIASxβ), SUMO2, and SUMO3. The increased gene expression resulted in higher levels of the respective proteins and changes in the pattern of sumoylated substrate proteins during the differentiation process. Similar to the HaCaT results, stratified human foreskin keratinocytes showed an upregulation of Ubc9 in the suprabasal layers. Lastly, abrogation of sumoylation by Gam1 expression severely disrupted normal HaCaT differentiation, consistent with an important role for sumoylation in the proper progression of this biological process. PMID:17164289

Cap analog and Potato virus A HC-Pro silencing suppressor improve GFP transient expression using an infectious virus vector in Nicotiana benthamiana.

PubMed

Tahmasebi, Amin-Alah; Afsharifar, Alireza

2017-06-01

Transient expression of proteins in plants has become a choice to facilitate recombinant protein production with its fast and easy application. On the other hand, host defensive mechanisms have been reported to reduce the efficiency of transient expression in plants. Hence, this study was designed to evaluate the effect of cap analog and Potato virus A helper component proteinase (PVA HC-Pro) on green fluorescent protein (GFP) expression efficiency. N . benthamiana leaves were inoculated with capped or un-capped RNA transcripts of a Turnip crinkle virus (TCV) construct containing a green fluorescent protein reporter gene (TCV-sGFP) in place of its coat protein (CP) ORF. PVA HC-Pro as a viral suppressor of RNA silencing was infiltrated in trans by Agrobacterium tumefaciens , increased the GFP foci diameter to six and even more cells in both capped and un capped treatments. The expression level of GFP in inoculated plants with TCV-sGFP transcript pre-infiltrated with PVA HC-Pro was 12.97-fold higher than the GFP accumulation level in pre-infiltrated leaves with empty plasmid (EP) control. Also, the yield of GFP in inoculated N. benthamiana plants with capped TCV-sGFP transcript pre-infiltrated with EP and PVA HC-Pro was 1.54 and 1.2-fold respectively, greater than the level of GFP expressed without cap analog application at 5 days post inoculation (dpi). In addition, the movement of TCV-sGFP was increased in some cells of inoculated leaves with capped transcripts. Results of this study indicated that PVA HC-Pro and mRNA capping can increase GFP expression and its cell to cell movement in N. benthamiana .

Attenuation of Ca2+ homeostasis, oxidative stress, and mitochondrial dysfunctions in diabetic rat heart: insulin therapy or aerobic exercise?

PubMed

da Silva, Márcia F; Natali, Antônio J; da Silva, Edson; Gomes, Gilton J; Teodoro, Bruno G; Cunha, Daise N Q; Drummond, Lucas R; Drummond, Filipe R; Moura, Anselmo G; Belfort, Felipe G; de Oliveira, Alessandro; Maldonado, Izabel R S C; Alberici, Luciane C

2015-07-15

We tested the effects of swimming training and insulin therapy, either alone or in combination, on the intracellular calcium ([Ca(2+)]i) homeostasis, oxidative stress, and mitochondrial functions in diabetic rat hearts. Male Wistar rats were separated into control, diabetic, or diabetic plus insulin groups. Type 1 diabetes mellitus was induced by streptozotocin (STZ). Insulin-treated groups received 1 to 4 UI of insulin daily for 8 wk. Each group was divided into sedentary or exercised rats. Trained groups were submitted to swimming (90 min/day, 5 days/wk, 8 wk). [Ca(2+)]i transient in left ventricular myocytes (LVM), oxidative stress in LV tissue, and mitochondrial functions in the heart were assessed. Diabetes reduced the amplitude and prolonged the times to peak and to half decay of the [Ca(2+)]i transient in LVM, increased NADPH oxidase-4 (Nox-4) expression, decreased superoxide dismutase (SOD), and increased carbonyl protein contents in LV tissue. In isolated mitochondria, diabetes increased Ca(2+) uptake, susceptibility to permeability transition pore (MPTP) opening, uncoupling protein-2 (UCP-2) expression, and oxygen consumption but reduced H2O2 release. Swimming training corrected the time course of the [Ca(2+)]i transient, UCP-2 expression, and mitochondrial Ca(2+) uptake. Insulin replacement further normalized [Ca(2+)]i transient amplitude, Nox-4 expression, and carbonyl content. Alongside these benefits, the combination of both therapies restored the LV tissue SOD and mitochondrial O2 consumption, H2O2 release, and MPTP opening. In conclusion, the combination of swimming training with insulin replacement was more effective in attenuating intracellular Ca(2+) disruptions, oxidative stress, and mitochondrial dysfunctions in STZ-induced diabetic rat hearts. Copyright © 2015 the American Physiological Society.

The theory of transient radiation of a charged particle in a waveguide with an anisotropic magnetodielectric filling

NASA Astrophysics Data System (ADS)

Gevorkyan, E. A.

2015-08-01

We have considered transient radiation of a charged particle that moves at a constant velocity perpendicularly to the axis of a regular waveguide filled with an anisotropic magnetodielectric medium. Wave equations and analytical expressions for transverse electric (TE) and transverse magnetic (TM) fields in the waveguide have been found. Energies of transient radiation of the particle moving in a rectangular waveguide have been determined. We have obtained conditions of occurrence, the frequency, and the energy of Vavilov-Cherenkov radiation.

High resolution imaging studies into the formation of laser-induced periodic surface structures.

PubMed

Kerr, N C; Clark, S E; Emmony, D C

1989-09-01

We report the results of an investigation into the formation mechanism of laser-induced ripple structures based on obtaining direct images of a surface while the transient heating induced by a KrF excimer laser is still present. These images reveal transient but well-defined periodic heating patterns which, if enough subsequent excimer pulses are incident on the surface, become permanently induced ripple structures. It is evident from these transient images that the surface heating is confined to the induced structures, thus strongly supporting the idea that at low fluences the ripples are formed by localizing surface melting.

alpha2-chimaerin, a Cdc42/Rac1 regulator, is selectively expressed in the rat embryonic nervous system and is involved in neuritogenesis in N1E-115 neuroblastoma cells.

PubMed

Hall, C; Michael, G J; Cann, N; Ferrari, G; Teo, M; Jacobs, T; Monfries, C; Lim, L

2001-07-15

Neuronal differentiation involves Rac and Cdc42 GTPases. alpha-Chimaerin, a Rac/Cdc42 regulator, occurs as alpha1- and alternatively spliced Src homology 2 (SH2) domain-containing alpha2-isoforms. alpha2-chimaerin mRNA was highly expressed in the rat embryonic nervous system, especially in early postmitotic neurons. alpha1-chimaerin mRNA was undetectable before embryonic day 16.5. Adult alpha2-chimaerin mRNA was restricted to neurons within specific brain regions, with highest expression in the entorhinal cortex. alpha2-chimaerin protein localized to neuronal perikarya, dendrites, and axons. The overall pattern of alpha2-chimaerin mRNA expression resembles that of cyclin-dependent kinase regulator p35 (CDK5/p35) which participates in neuronal differentiation and with which chimaerin interacts. To determine whether alpha2-chimaerin may have a role in neuronal differentiation and the relevance of the SH2 domain, the morphological effects of both chimaerin isoforms were investigated in N1E-115 neuroblastoma cells. When plated on poly-lysine, transient alpha2-chimaerin but not alpha1-chimaerin transfectants formed neurites. Permanent alpha2-chimaerin transfectants generated neurites whether or not they were stimulated by serum starvation, and many cells were enlarged. Permanent alpha1-chimaerin transfectants displayed numerous microspikes and contained F-actin clusters, a Cdc42-phenotype, but generated few neurites. In neuroblastoma cells, alpha2-chimaerin was predominantly soluble with some being membrane-associated, whereas alpha1-chimaerin was absent from the cytosol, being membrane- and cytoskeleton-associated, paralleling their subcellular distribution in brain. Transient transfection with alpha2-chimaerin mutated in the SH2 domain (N94H) generated an alpha1-chimaerin-like phenotype, protein partitioned in the particulate fraction, and in NGF-stimulated pheochromocytoma cell line 12 (PC12) cells, neurite formation was inhibited. These results indicate a role for alpha2-chimaerin in morphological differentiation for which its SH2 domain is vital.

Gene delivery to the neurulating embryo during culture

EPA Science Inventory

Modulating expression of specific genes during embryogenesis will help elucidate their role in development. Transient overexpression of specific genes can be accomplished by adding additional copies, or else antisense transcripts can be used to block expression. Manipulation of g...

Transient hypothyroidism after iodine-131 therapy for Grave's disease.

PubMed

Gómez, N; Gómez, J M; Orti, A; Gavaldà, L; Villabona, C; Leyes, P; Soler, J

1995-09-01

We studied 355 patients with Grave's disease to characterize transient hypothyroidism and its prognostic value following 131I therapy. The patients received therapeutic 131I treatment as follows: 333 received a dose < 10 mCi (6.6 +/- 1.9 mCi) and 22 received a dose > 10 mCi (12.8 +/- 2.9 mCi). Diagnosis of transient hypothyroidism was based on low T4, regardless of TSH within the first year after 131I followed by recovery of T4 and normal TSH. After administration of < 10 mCi 131I, 40 patients developed transient hypothyroidism during the first year; transient hypothyroidism was symptomatic in 15. There was no transient hypothyroidism after high doses (> 10 mCi) of 131I. Iodine-131 uptake > 70% at 2 hr before treatment was a risk factor for developing transient hypothyroidism (Odds ratio 2.8, 95% confidence interval 0.9-9.4). At diagnosis of transient hypothyroidism, basal TSH levels were high (51%), normal (35%) or low (14%); therefore, the transient hypothyroidism was not centralized. If hypothyroidism developed during the first 6 mo after basal TSH > 45 mU/liter ruled out transient hypothyroidism. The development of transient hypothyroidism and its hormonal pattern did not influence long-term thyroid function. Since no prognostic factors reliably predicted transient hypothyroidism before 131I or at the time of diagnosis, if hypothyroidism appears within the first months after 131I, the reevaluation of thyroid function later is warranted to avoid unnecessary chronic replacement therapy.

Temperature-Centric Evaluation of Sensor Transients

NASA Astrophysics Data System (ADS)

Ayhan, Tuba; Muezzinoglu, Kerem; Vergara, Alexander; Yalcin, Mustak

2011-09-01

Controllable sensing conditions provide the means for diversifying sensor response and achieving better selectivity. Modulating the sensing layer temperature of metal-oxide sensors is a popular method for multiplexing the limited number of sensing elements that can be employed in a practical array. Time limitations in many applications, however, cannot tolerate an ad-hoc, one-size-fits-all modulation pattern. When the response pattern is itself non-stationary, as in the transient phase, a temperature program also becomes infeasible. We consider the problem of determining and tuning into a fixed optimum temperature in a sensor array. For this purpose, we present an empirical analysis of the temperature's role on the performance of a metal-oxide gas sensor array in the identification of odorants along the response transient. We show that the optimal temperature in this sense depends heavily on the selection of (i) the set of candidate analytes, (ii) the time-window of the analysis, (iii) the feature extracted from the sensor response, and (iv) the computational identification method used.

Development and characterization of reduced graphene oxide films for transient electronics

NASA Astrophysics Data System (ADS)

Sheikh, Rasel; Bhatkar, Omkar; Smith, David; Rizvi, Reza

2018-03-01

Emerging interests in hardware security as well as environmental concerns have given rise to the field of transient or temporary electronics, which can be decommissioned by an external stimulus with minimal impact to the surrounding environment. In this study, an all graphene based film is produced by a one-step deposition process. The conversion of graphene oxide (GO) to reduced graphene oxide (rGO) depends on an interfacial reduction reaction. Control of processing conditions such as the underlying substrate, pH of GO and the film drying environment results in an ability to tailor the internal architecture of the films and their electronic properties. Furthermore, the ability to create masks for selective reduction of GO during deposition was also demonstrated, which was used to create intricate yet well-defined patterns and connections required in electronic circuits and devices. All graphene based freestanding films with selectively reduced GO were used in transient electronics application as circuitry and RFID tag patterns.

Suppression of transient receptor potential melastatin 4 expression promotes conversion of endothelial cells into fibroblasts via transforming growth factor/activin receptor-like kinase 5 pathway.

PubMed

Echeverría, Cesar; Montorfano, Ignacio; Cabello-Verrugio, Claudio; Armisén, Ricardo; Varela, Diego; Simon, Felipe

2015-05-01

To study whether transient receptor potential melastatin 4 (TRPM4) participates in endothelial fibrosis and to investigate the underlying mechanism. Primary human endothelial cells were used and pharmacological and short interfering RNA-based approaches were used to test the transforming growth factor beta (TGF-β)/activin receptor-like kinase 5 (ALK5) pathway participation and contribution of TRPM7 ion channel. Suppression of TRPM4 expression leads to decreased endothelial protein expression and increased expression of fibrotic and extracellular matrix markers. Furthermore, TRPM4 downregulation increases intracellular Ca levels as a potential condition for fibrosis. The underlying mechanism of endothelial fibrosis shows that inhibition of TRPM4 expression induces TGF-β1 and TGF-β2 expression, which act through their receptor, ALK5, and the nuclear translocation of the profibrotic transcription factor smad4. TRPM4 acts to maintain endothelial features and its loss promotes fibrotic conversion via TGF-β production. The regulation of TRPM4 levels could be a target for preserving endothelial function during inflammatory diseases.

A Transient Expression of Prospero Promotes Cell Cycle Exit of Drosophila Postembryonic Neurons through the Regulation of Dacapo

PubMed Central

Colonques, Jordi; Ceron, Julian; Reichert, Heinrich; Tejedor, Francisco J.

2011-01-01

Cell proliferation, specification and terminal differentiation must be precisely coordinated during brain development to ensure the correct production of different neuronal populations. Most Drosophila neuroblasts (NBs) divide asymmetrically to generate a new NB and an intermediate progenitor called ganglion mother cell (GMC) which divides only once to generate two postmitotic cells called ganglion cells (GCs) that subsequently differentiate into neurons. During the asymmetric division of NBs, the homeodomain transcription factor PROSPERO is segregated into the GMC where it plays a key role as cell fate determinant. Previous work on embryonic neurogenesis has shown that PROSPERO is not expressed in postmitotic neuronal progeny. Thus, PROSPERO is thought to function in the GMC by repressing genes required for cell-cycle progression and activating genes involved in terminal differentiation. Here we focus on postembryonic neurogenesis and show that the expression of PROSPERO is transiently upregulated in the newly born neuronal progeny generated by most of the larval NBs of the OL and CB. Moreover, we provide evidence that this expression of PROSPERO in GCs inhibits their cell cycle progression by activating the expression of the cyclin-dependent kinase inhibitor (CKI) DACAPO. These findings imply that PROSPERO, in addition to its known role as cell fate determinant in GMCs, provides a transient signal to ensure a precise timing for cell cycle exit of prospective neurons, and hence may link the mechanisms that regulate neurogenesis and those that control cell cycle progression in postembryonic brain development. PMID:21552484

Regulation of early gene expression from the bovine papillomavirus genome in transiently transfected C127 cells.

PubMed Central

Szymanski, P; Stenlund, A

1991-01-01

Expression of bovine papillomavirus (BPV) early gene products is required for viral DNA replication and establishment of the transformed phenotype. By the use of a highly efficient electroporation system, we have examined for the first time the transcriptional activity of BPV promoters in their natural genomic context in a replication-permissive cell line. We have determined that a qualitatively distinct stage of transcription is not detectable prior to DNA replication in transiently transfected cells. This suggests that the transcriptional activity of the BPV genome in stably transformed cells represents the early stage of BPV gene expression. Quantitative differences in promoter activity between transiently transfected and stably transformed cells suggest that subtle changes in gene expression may control progression of the viral life cycle. Deletion analysis demonstrated that the E2 transactivator protein stimulates all of the early promoters through sequences located in the upstream regulatory region. This E2-dependent enhancer was found to be highly redundant, and particular E2 binding sites did not display a preference for particular promoters. Despite this dependence on a common cis-acting sequence, the various promoters displayed different sensitivities to the E2 transactivator. The findings that E2 regulates all promoters and, with the exception of the E2 repressors, that no other known viral gene product appears to affect transcription indicate that the E2 system functions as the master regulator of BPV early gene expression. Images PMID:1656065

Properties of voltage-activated Na+ and K+ currents in mouse hippocampal glial cells in situ and after acute isolation from tissue slices.

PubMed

Steinhäuser, C; Kressin, K; Kuprijanova, E; Weber, M; Seifert, G

1994-10-01

In the present study, we were interested in a quantitative analysis of voltage-activated channels in a subpopulation of hippocampal glial cells, termed "complex" cells. The patch-clamp technique in the whole-cell mode was applied to identified cells in situ and to glial cells acutely isolated from tissue slices. The outward current was composed of two components: a sustained and a transient current. The transient K+ channel had electrophysiological and pharmacological properties resembling those of the channel through which the A-currents pass. In addition, this glial A-type current possessed a significant Ca2+ dependence. The current parameters determined in situ or in isolated cells corresponded well. Due to space clamp problems in situ, properties of voltage-dependent Na+ currents were only analysed in suspended glial cells. The tetrodotoxin (TTX) sensitivity and the stationary and kinetic characteristics of this current were similar to corresponding properties of hippocampal neurons. These quantitative data demonstrate that at an early postnatal stage of central nervous system maturation, glial cells in situ express a complex pattern of voltage-gated ion channels. The results are compared to findings in other preparations and the possible consequences of transmitter-mediated channel modulation in glial cells are discussed.

Ultrasonic transient bounded-beam propagation in a solid cylinder waveguide embedded in a solid medium.

PubMed

Laguerre, Laurent; Grimault, Anne; Deschamps, Marc

2007-04-01

A semianalytical solution alternative and complementary to modal technique is presented to predict and interpret the ultrasonic pulsed-bounded-beam propagation in a solid cylinder embedded in a solid matrix. The spectral response to an inside axisymmetric velocity source of longitudinal and transversal cylindrical waves is derived from Debye series expansion of the embedded cylinder generalized cylindrical reflection/transmission coefficients. So, the transient guided wave response, synthesized by inverse double Fourier-Bessel transform, is expressed as a combination of the infinite medium contribution, longitudinal, transversal, and coupled longitudinal and transversal waveguide sidewall interactions. Simulated (f, 1/lambdaz) diagrams show the influence of the number of waveguide sidewall interactions to progressively recover dispersion curves. Besides, they show the embedding material filters specific signal portions by concentrating the propagating signal in regions where phase velocity is closer to phase velocity in steel. Then, simulated time waveforms using broadband high-frequency excitation show that signal leading portions exhibit a similar periodical pattern, for both free and embedded waveguides. Debye series-based interpretation shows that double longitudinal/transversal and transversal/longitudinal conversions govern the time waveform leading portion as well as the radiation attenuation in the surrounding cement grout. Finally, a methodology is deduced to minimize the radiation attenuation for the long-range inspection of embedded cylinders.

Transient and permanent changes in DNA methylation patterns in inorganic arsenic-mediated epithelial-to-mesenchymal transition

PubMed Central

Eckstein, Meredith; Rea, Matthew; Fondufe-Mittendorf, Yvonne N.

2017-01-01

Chronic low dose inorganic arsenic exposure causes cells to take on an epithelial-to-mesenchymal phenotype, which is a crucial process in carcinogenesis. Inorganic arsenic is not a mutagen and thus epigenetic alterations have been implicated in this process. Indeed, during the epithelial-to-mesenchymal transition, morphologic changes to cells correlate with changes in chromatin structure and gene expression, ultimately driving this process. However, studies on the effects of inorganic arsenic exposure/withdrawal on the epithelial-to-mesenchymal transition and the impact of epigenetic alterations in this process are limited. In this study we used high-resolution microarray analysis to measure the changes in DNA methylation in cells undergoing inorganic arsenic-induced epithelial-to-mesenchymal transition, and on the reversal of this process, after removal of the inorganic arsenic exposure. We found that cells exposed to chronic, low-dose inorganic arsenic exposure showed 30,530 sites were differentially methylated, and with inorganic arsenic withdrawal several differential methylated sites were reversed, albeit not completely. Furthermore, these changes in DNA methylation mainly correlated with changes in gene expression at most sites tested but not at all. This study suggests that DNA methylation changes on gene expression are not clear-cut and provide a platform to begin to uncover the relationship between DNA methylation and gene expression, specifically within the context of inorganic arsenic treatment. PMID:28336213

Expression analysis and functional characterization of a novel cold-responsive gene CbCOR15a from Capsella bursa-pastoris.

PubMed

Zhou, Mingqi; Wu, Lihua; Liang, Jing; Shen, Chen; Lin, Juan

2012-05-01

The cold-responsive (COR) genes involved in C-repeat binding factor signaling pathway function essentially in cold acclimation of higher plants. A novel COR gene CbCOR15a from shepherd's purse (Capsella bursa-pastoris) was predicted to be a homolog of COR15 in Arabidopsis. The analysis of tissue specific expression pattern as well as characterization of the CbCOR15a promoter revealed that the expression of CbCOR15a was induced by coldness not only in leaves and stem but also in roots. Sequence analysis showed that a 909 bp promoter region of CbCOR15a contained two CRT/DRE elements, two ABRE elements, one auxin-responsive TGA-element and one MeJA-responsive CGTCA-motif. In young seedlings the expression of CbCOR15a could be apparently increased by SA, ABA, MeJA and IAA, and transiently increased by GA(3) accompanied by obvious feedback suppression. According to the altered physiological index values in tobacco under cold treatments, the overexpression of CbCOR15a significantly increased the cold tolerance of transgenic tobacco plants. It can be suggested that CbCOR15a was involved in cold response of Capsella bursa-pastoris associated with SA, ABA, MeJA, IAA and GA(3) regulation and confers enhanced cold acclimation in transgenic plants.

Pattern of calbindin-D28k and calretinin immunoreactivity in the brain of Xenopus laevis during embryonic and larval development.

PubMed

Morona, Ruth; González, Agustín

2013-01-01

The present study represents a detailed spatiotemporal analysis of the localization of calbindin-D28k (CB) and calretinin (CR) immunoreactive structures in the brain of Xenopus laevis throughout development, conducted with the aim to correlate the onset of the immunoreactivity with the development of compartmentalization of distinct subdivisions recently identified in the brain of adult amphibians and primarily highlighted when analyzed within a segmental paradigm. CR and CB are expressed early in the brain and showed a progressively increasing expression throughout development, although transient expression in some neuronal subpopulations was also noted. Common and distinct characteristics in Xenopus, as compared with reported features during development in the brain of mammals, were observed. The development of specific regions in the forebrain such as the olfactory bulbs, the components of the basal ganglia and the amygdaloid complex, the alar and basal hypothalamic regions, and the distinct diencephalic neuromeres could be analyzed on the basis of the distinct expression of CB and CR in subregions. Similarly, the compartments of the mesencephalon and the main rhombencephalic regions, including the cerebellum, were differently highlighted by their specific content in CB and CR throughout development. Our results show the usefulness of the analysis of the distribution of these proteins as a tool in neuroanatomy to interpret developmental aspects of many brain regions. Copyright © 2012 Wiley Periodicals, Inc.

Nitric oxide signaling in the development and evolution of language and cognitive circuits.

PubMed

Funk, Owen H; Kwan, Kenneth Y

2014-09-01

The neocortex underlies not only remarkable motor and sensory capabilities, but also some of our most distinctly human cognitive functions. The emergence of these higher functions during evolution was accompanied by structural changes in the neocortex, including the acquisition of areal specializations such as Broca's speech and language area. The study of these evolutionary mechanisms, which likely involve species-dependent gene expression and function, represents a substantial challenge. These species differences, however, may represent valuable opportunities to understand the molecular underpinnings of neocortical evolution. Here, we discuss nitric oxide signaling as a candidate mechanism in the assembly of neocortical circuits underlying language and higher cognitive functions. This hypothesis was based on the highly specific mid-fetal pattern of nitric oxide synthase 1 (NOS1, previously nNOS) expression in the pyramidal (projection) neurons of two human neocortical areas respectively involved in speech and language, and higher cognition; the frontal operculum (FOp) and the anterior cingulate cortex (ACC). This expression is transiently present during mid-gestation, suggesting that NOS1 may be involved in the development of these areas and the assembly of their neural circuits. As no other gene product is known to exhibit such exquisite spatiotemporal expression, NOS1 represents a remarkable candidate for these functions. Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Decreased akt activity is associated with activation of forkhead transcription factor after transient forebrain ischemia in gerbil hippocampus.

PubMed

Kawano, Takayuki; Morioka, Motohiro; Yano, Shigetoshi; Hamada, Jun-Ichiro; Ushio, Yukitaka; Miyamoto, Eishichi; Fukunaga, Kohji

2002-08-01

The authors recently reported that sodium orthovanadate rescues cells from delayed neuronal death in gerbil hippocampus after transient forebrain ischemia through phosphatidylinositol 3-kinase-protein kinase B (Akt) pathway (Kawano et al., 2001). In the current study, they demonstrated that the activation of FKHR, a Forkhead transcription factor and a substrate for Akt, preceded delayed neuronal death in CA1 regions after transient forebrain ischemia. Adult Mongolian gerbils were subjected to 5-minute forebrain ischemia. Immunoblotting analysis with anti-phospho-FKHR antibody showed that phosphorylation of FKHR at serine-256 in the CA1 region decreased immediately after and 0.5 and 1 hour after reperfusion. The dephosphorylation of FKHR was correlated with the decreased Akt activity. Intracerebroventricular injection of orthovanadate 30 minutes before ischemia inhibited dephosphorylation of FKHR after reperfusion, and blocked delayed neuronal death in the CA1 region. Gel mobility shift analysis using nuclear extracts from the CA1 region prepared immediately after reperfusion revealed increases in DNA binding activity for the FKHR-responsive element on the Fas ligand promoter. The orthovanadate injection administered before ischemia inhibited its binding activity. Two days after reperfusion, expression of Fas ligand increased in the CA1 region and the orthovanadate injection inhibited this increased expression. These results suggest that the inactivation of Akt results in the activation of FKHR and, in turn, relates to the expression of Fas ligand in the CA1 region after transient forebrain ischemia.

A computational analysis of the impact of the transient genetic imbalance on compartmentalized gene expression during sporulation in Bacillus subtilis.

PubMed

Iber, Dagmar

2006-06-30

Sporulation in Bacillus subtilis serves as paradigm for the development of two different cell types (mother cell and prespore) from a single cell. Differential gene expression is achieved by restricting the activation of the key transcription factor sigmaF to the smaller prespore. By use of a combination of mathematical and experimental techniques we have recently shown that the volume difference determines cell fate and that the accumulation of the phosphatase SpoIIE on the asymmetrically placed septum is sufficient for prespore-specific sigmaF activation. Since compartmentalized gene expression is still obtained when SpoIIE cannot accumulate on the septum a number of alternative mechanisms have been proposed. These mechanisms focus on the difference in gene content between mother cell and prespore immediately after septation. Here the computational model is employed to show that under physiological conditions the transient genetic imbalance is unlikely to affect the septation-dependent release of sigmaF. The duration of the transient genetic imbalance is too short for the degradation of SpoIIAB to have an impact on the release of sigmaF. Moreover, the existence of an elusive IIE inhibitor, which has been proposed to become depleted in the prespore because of the transient genetic imbalance, is shown to be inconsistent with available experimental data. We conclude that the volume difference between the two compartments is the main determinant of cell fate.

Unique processing during a period of high excitation/inhibition balance in adult-born neurons.

PubMed

Marín-Burgin, Antonia; Mongiat, Lucas A; Pardi, M Belén; Schinder, Alejandro F

2012-03-09

The adult dentate gyrus generates new granule cells (GCs) that develop over several weeks and integrate into the preexisting network. Although adult hippocampal neurogenesis has been implicated in learning and memory, the specific role of new GCs remains unclear. We examined whether immature adult-born neurons contribute to information encoding. By combining calcium imaging and electrophysiology in acute slices, we found that weak afferent activity recruits few mature GCs while activating a substantial proportion of the immature neurons. These different activation thresholds are dictated by an enhanced excitation/inhibition balance transiently expressed in immature GCs. Immature GCs exhibit low input specificity that switches with time toward a highly specific responsiveness. Therefore, activity patterns entering the dentate gyrus can undergo differential decoding by a heterogeneous population of GCs originated at different times.

Constitutive Activity among Orphan Class-A G Protein Coupled Receptors.

PubMed

Martin, Adam L; Steurer, Michael A; Aronstam, Robert S

2015-01-01

The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention.

Constitutive Activity among Orphan Class-A G Protein Coupled Receptors

PubMed Central

Martin, Adam L.; Steurer, Michael A.; Aronstam, Robert S.

2015-01-01

The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention. PMID:26384023

Four RNA families with functional transient structures

PubMed Central

Zhu, Jing Yun A; Meyer, Irmtraud M

2015-01-01

Protein-coding and non-coding RNA transcripts perform a wide variety of cellular functions in diverse organisms. Several of their functional roles are expressed and modulated via RNA structure. A given transcript, however, can have more than a single functional RNA structure throughout its life, a fact which has been previously overlooked. Transient RNA structures, for example, are only present during specific time intervals and cellular conditions. We here introduce four RNA families with transient RNA structures that play distinct and diverse functional roles. Moreover, we show that these transient RNA structures are structurally well-defined and evolutionarily conserved. Since Rfam annotates one structure for each family, there is either no annotation for these transient structures or no such family. Thus, our alignments either significantly update and extend the existing Rfam families or introduce a new RNA family to Rfam. For each of the four RNA families, we compile a multiple-sequence alignment based on experimentally verified transient and dominant (dominant in terms of either the thermodynamic stability and/or attention received so far) RNA secondary structures using a combination of automated search via covariance model and manual curation. The first alignment is the Trp operon leader which regulates the operon transcription in response to tryptophan abundance through alternative structures. The second alignment is the HDV ribozyme which we extend to the 5′ flanking sequence. This flanking sequence is involved in the regulation of the transcript's self-cleavage activity. The third alignment is the 5′ UTR of the maturation protein from Levivirus which contains a transient structure that temporarily postpones the formation of the final inhibitory structure to allow translation of maturation protein. The fourth and last alignment is the SAM riboswitch which regulates the downstream gene expression by assuming alternative structures upon binding of SAM. All transient and dominant structures are mapped to our new alignments introduced here. PMID:25751035

Four RNA families with functional transient structures.

PubMed

Zhu, Jing Yun A; Meyer, Irmtraud M

2015-01-01

Protein-coding and non-coding RNA transcripts perform a wide variety of cellular functions in diverse organisms. Several of their functional roles are expressed and modulated via RNA structure. A given transcript, however, can have more than a single functional RNA structure throughout its life, a fact which has been previously overlooked. Transient RNA structures, for example, are only present during specific time intervals and cellular conditions. We here introduce four RNA families with transient RNA structures that play distinct and diverse functional roles. Moreover, we show that these transient RNA structures are structurally well-defined and evolutionarily conserved. Since Rfam annotates one structure for each family, there is either no annotation for these transient structures or no such family. Thus, our alignments either significantly update and extend the existing Rfam families or introduce a new RNA family to Rfam. For each of the four RNA families, we compile a multiple-sequence alignment based on experimentally verified transient and dominant (dominant in terms of either the thermodynamic stability and/or attention received so far) RNA secondary structures using a combination of automated search via covariance model and manual curation. The first alignment is the Trp operon leader which regulates the operon transcription in response to tryptophan abundance through alternative structures. The second alignment is the HDV ribozyme which we extend to the 5' flanking sequence. This flanking sequence is involved in the regulation of the transcript's self-cleavage activity. The third alignment is the 5' UTR of the maturation protein from Levivirus which contains a transient structure that temporarily postpones the formation of the final inhibitory structure to allow translation of maturation protein. The fourth and last alignment is the SAM riboswitch which regulates the downstream gene expression by assuming alternative structures upon binding of SAM. All transient and dominant structures are mapped to our new alignments introduced here.

Developing grasshopper neurons show variable levels of guanylyl cyclase activity on arrival at their targets.

PubMed

Ball, E E; Truman, J W

1998-04-27

The ability of certain grasshopper neurons to respond to exogenously applied donors of nitric oxide (NO) by producing cyclic GMP (cGMP) depends on their developmental state. ODQ, a selective blocker of NO-sensitive guanylyl cyclase, blocks cGMP production at 10(-5) M, thus confirming the nature of the response. Experiments in which the distal axon is separated from its proximal stump before application of an NO donor show that guanylyl cyclase is distributed uniformly throughout the neuron. In the locust abdomen, where segments are formed sequentially, the pattern of guanylyl cyclase up-regulation is predictable and sequential from anterior to posterior. There are two patterns of innervation by cGMP-expressing motor neurons. In the first, typified by muscle 187, an innervating neuron begins to be NO responsive on arrival at its muscle and continues to be so over most of the remainder of embryonic development, including the formation of motor end plates. In the second, typified by a neuron innervating muscle 191, the neuron extends well along the muscle, apparently laying down a number of sites of contact with it, before it becomes NO responsive. In both patterns, however, NO responsiveness marks the neuron's transition from growth cone elongation to the production of lateral branches. Individual muscles receive innervation from multiple motor neurons, some of which express transient NO sensitivity during development and others which do not. With the exception of the leg motor neuron SETi, the first motor neuron to reach any muscle is usually not NO responsive. We suggest that cGMP plays a role in, or reflects, the early stages of communication between a target and specific innervating neurons.

Isolation of a citrus promoter specific for reproductive organs and its functional analysis in isolated juice sacs and tomato.

PubMed

Sorkina, Alina; Bardosh, Gabriel; Liu, Yong-Zhong; Fridman, Ifat; Schlizerman, Ludmila; Zur, Naftali; Or, Etti; Goldschmidt, Eliezer E; Blumwald, Eduardo; Sadka, Avi

2011-09-01

While searching for genes expressed in acid lemon but not in acidless lime pulp, we isolated clone Cl111 which showed the following expression phenotypes: (1) while it was expressed in the ovaries in both varieties, its mRNA was detected only in the pulp of the acid fruit, (2) no or very low expression of the gene was detected in vegetative organs. These expression patterns suggested that Cl111 is an ovary- and pulp-specific gene. The ability of ~2-kb fragments upstream of the transcription start site of the lemon and lime genes to confer reporter-gene activity was investigated by transient expression in isolated juice vesicles of both varieties. Whereas Cl111 promoter from lemon showed faint activity in lemon and lime juice vesicles, no activity was evident with the lime promoter. The activities of the 2-kb fragments and their delimited fragments were further investigated in tomato. The results indicated that the promoters were active in a manner similar to that in acid lemon and acidless lime: the lemon promoter generated activity in the fruit endocarp, analogous to citrus fruit pulp. The delimitation analyses identified an expression-conferring region which, in the lemon promoter, contained a sequence homologous to a fruit-specific element of the melon cucumisin gene. Another region, which reduced promoter activity, contained an I-Box-like sequence, identified as a fruit-specific negative element. Taken together, Cl111 promoter was confirmed to be pulp- and flower-specific. Differences in the expression of Cl111 between the two varieties could be attributable to changes in the gene promoter region.

Amyloid beta peptide as a physiological modulator of neuronal 'A'-type K+ current.

PubMed

Plant, Leigh D; Webster, Nicola J; Boyle, John P; Ramsden, Martin; Freir, Darragh B; Peers, Chris; Pearson, Hugh A

2006-11-01

Control of neuronal spiking patterns resides, in part, in the type and degree of expression of voltage-gated K(+) channel subunits. Previous studies have revealed that soluble forms of the Alzheimer's disease associated amyloid beta protein (Abeta) can increase the 'A'-type current in neurones. In this study, we define the molecular basis for this increase and show that endogenous production of Abeta is important in the modulation of Kv4.2 and Kv4.3 subunit expression in central neurones. A-type K(+) currents, and Kv4.2 and Kv4.3 subunit expression, were transiently increased in cerebellar granule neurones by the 1-40 and 1-42 forms of Abeta (100nM, 2-24h). Currents through recombinant Kv4.2 channels expressed in HEK293 cells were increased in a similar fashion to those through the native channels. Increases in 'A'-type current could be prevented by the use of cycloheximide and brefeldin A, indicating that protein expression and trafficking processes were altered by Abeta, rather than protein degredation. Endogenous Abeta production in cerebellar granule neurones was blocked using inhibitors of either gamma- or beta-secretase and resulted in decreased K(+) current. Crucially this could be prevented by co-application of exogenous Abeta (1nM), however, no change in Kv4.2 or Kv4.3 subunit expression occurred. These data show that Abeta is a modulator of Kv4 subunit expression in neurones at both the functional and the molecular level. Thus Abeta is not only involved in Alzheimer pathology, but is also an important physiological regulator of ion channel expression and hence neuronal excitability.

Emergence delirium with transient associative agnosia and expressive aphasia reversed by flumazenil in a pediatric patient.

PubMed

Drobish, Julie K; Kelz, Max B; DiPuppo, Patricia M; Cook-Sather, Scott D

2015-06-01

Multiple factors may contribute to the development of emergence delirium in a child. We present the case of a healthy 12-year-old girl who received preoperative midazolam with the desired anxiolytic effect, underwent a brief general anesthetic, and then exhibited postoperative delirium, consisting of a transient associative agnosia and expressive aphasia. Administration of flumazenil led to immediate and lasting resolution of her symptoms. We hypothesize that γ-aminobutyric acid type A receptor-mediated effects, most likely related to an atypical offset of midazolam, are an important subset of emergence delirium that is amenable to pharmacologic therapy with flumazenil.

Emergence Delirium with Transient Associative Agnosia and Expressive Aphasia Reversed by Flumazenil in a Pediatric Patient

PubMed Central

Drobish, Julie K.; Kelz, Max B.; DiPuppo, Patricia M.; Cook-Sather, Scott D.

2014-01-01

Multiple factors may contribute to the development of emergence delirium in a child. We present the case of a healthy 12-year-old girl who received preoperative midazolam with the desired anxiolytic effect, underwent a brief general anesthetic, and then exhibited postoperative delirium, consisting of a transient associative agnosia and expressive aphasia. Administration of flumazenil led to immediate and lasting resolution of her symptoms. We hypothesize that γ-aminobutyric acid type A receptor-mediated effects, most likely related to an atypical offset of midazolam, are an important subset of emergence delirium that is amenable to pharmacologic therapy with flumazenil. PMID:26035220

Analysis in temporal regime of dispersive invisible structures designed from transformation optics

NASA Astrophysics Data System (ADS)

Gralak, B.; Arismendi, G.; Avril, B.; Diatta, A.; Guenneau, S.

2016-03-01

A simple invisible structure made of two anisotropic homogeneous layers is analyzed theoretically in temporal regime. The frequency dispersion is introduced and analytic expression of the transient part of the field is derived for large times when the structure is illuminated by a causal excitation. This expression shows that the limiting amplitude principle applies with transient fields decaying as the power -3 /4 of the time. The quality of the cloak is then reduced at short times and remains preserved at large times. The one-dimensional theoretical analysis is supplemented with full-wave numerical simulations in two-dimensional situations which confirm the effect of dispersion.

Effects of plant density on recombinant hemagglutinin yields in an Agrobacterium-mediated transient gene expression system using Nicotiana benthamiana plants.

PubMed

Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

2017-08-01

Agrobacterium-mediated transient expression systems enable plants to rapidly produce a wide range of recombinant proteins. To achieve economically feasible upstream production and downstream processing, it is beneficial to obtain high levels of two yield-related quantities of upstream production: recombinant protein content per fresh mass of harvested biomass (g gFM -1 ) and recombinant protein productivity per unit area-time (g m -2 /month). Here, we report that the density of Nicotiana benthamiana plants during upstream production had significant impacts on the yield-related quantities of recombinant hemagglutinin (HA). The two quantities were smaller at a high plant density of 400 plants m -2 than at a low plant density of 100 plants m -2 . The smaller quantities at the high plant density were attributed to: (i) a lower HA content in young leaves, which usually have high HA accumulation potentials; (ii) a lower biomass allocation to the young leaves; and (iii) a high area-time requirement for plants. Thus, plant density is a key factor for improving upstream production in Agrobacterium-mediated transient expression systems. Biotechnol. Bioeng. 2017;114: 1762-1770. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Actions of bis(7)-tacrine and tacrine on transient potassium current in rat DRG neurons and potassium current mediated by K(V)4.2 expressed in Xenopus oocyte.

PubMed

Li, Xiang-Yuan; Zhang, Jian; Dai, Jia-Pei; Liu, Xiang-Ming; Li, Zhi-Wang

2010-03-08

Bis(7)-tacrine [bis(7)-tetrahydroaminacrine] is a dimeric AChE inhibitor derived from tacrine with a potential to treat Alzheimer's disease. Actions of bis(7)-tacrine on ligand-gated ion channels and voltage-gated cation channels have been identified on neurons of both central and peripheral nervous systems. In the present study, the effect of bis(7)-tacrine was investigated on the K(V)4.2 encoded potassium currents expressed in Xenopus oocytes and the transient A-type potassium current (I(K(A))) on rat DRG neurons. Bis(7)-tacrine suppressed recombinant Kv4.2 potassium channels in a concentration-dependent manner, with IC(50) value of 0.53+/-0.13 muM. Tacrine also inhibited Kv4.2 channels, but with a much lower potency (IC(50) 74+/-15 muM).The possible mechanisms underlying the inhibition on potassium currents by bis(7)-tacrine/tacrine could be that inactivation of the transient potassium currents was accelerated and recovery of the native or Kv4.2 expressed potassium currents was suppressed by bis(7)-tacrine/tacrine. Copyright 2010 Elsevier B.V. All rights reserved.

Local upregulation of transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 ion channels in rectosigmoid deep infiltrating endometriosis

PubMed Central

Bohonyi, Noémi; Pohóczky, Krisztina; Szalontai, Bálint; Perkecz, Anikó; Kovács, Krisztina; Kajtár, Béla; Orbán, Lajos; Varga, Tamás; Szegedi, Sarolta; Bódis, József; Koppán, Miklós

2017-01-01

Transient Receptor Potential Vanilloid 1 (TRPV1) and Transient Receptor Potential Ankyrin 1 (TRPA1) expressed mainly by primary sensory neurons function as major nociceptive integrators. They are also present on the rat endometrium in an oestrogen-regulated manner. TRPV1 is upregulated in peritoneal and ovarian endometriosis patients, but there is no information about TRPA1 and their pathophysiological significances. In this study, patients undergoing laparoscopic surgery were investigated: severe dysmenorrhoea due to rectosigmoid deep infiltrating endometriosis (n = 15), uterine fibroid-induced moderate dysmenorrhoea (n = 7) and tubal infertility with no pain (n = 6). TRPA1 and TRPV1 mRNA and protein expressions were determined by quantitative polymerase chain reaction and semi-quantitative immunohistochemistry from the endometrium samples taken by curettage. Results were correlated with the clinical characteristics including pain intensity. TRPA1 and TRPV1 receptors were expressed in the healthy human endometrium at mRNA and protein levels. Sparse, scattered cytoplasmic TRPA1 and TRPV1 immunopositivities were found in the stroma and epithelial layers. We detected upregulated mRNA levels in deep infiltrating endometriosis lesions, and TRPV1 gene expression was also elevated in autocontrol endometrium of deep infiltrating endometriosis patients. Histological scoring revealed significant TRPA1 and TRPV1 difference between deep infiltrating endometriosis stroma and epithelium, and in deep infiltrating endometriosis epithelium compared to control samples. Besides, we measured elevated stromal TRPV1 immunopositivity in deep infiltrating endometriosis. Stromal TRPA1 and TRPV1 immunoreactivities strongly correlated with dysmenorrhoea severity, as well TRPV1 expression on ectopic epithelial cells and macrophages with dyspareunia. Epithelial TRPA1 and stromal TRPV1 immunopositivity also positively correlated with dyschezia severity. We provide the first evidence for the presence of non-neuronal TRPA1 receptor in the healthy human endometrium and confirm the expression of TRPV1 channels. Their upregulations in rectosigmoid deep infiltrating endometriosis lesions and correlations with pain intensity suggest potential roles in pathophysiological mechanisms of the disease. PMID:28478727

Local upregulation of transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 ion channels in rectosigmoid deep infiltrating endometriosis.

PubMed

Bohonyi, Noémi; Pohóczky, Krisztina; Szalontai, Bálint; Perkecz, Anikó; Kovács, Krisztina; Kajtár, Béla; Orbán, Lajos; Varga, Tamás; Szegedi, Sarolta; Bódis, József; Helyes, Zsuzsanna; Koppán, Miklós

2017-01-01

Transient Receptor Potential Vanilloid 1 (TRPV1) and Transient Receptor Potential Ankyrin 1 (TRPA1) expressed mainly by primary sensory neurons function as major nociceptive integrators. They are also present on the rat endometrium in an oestrogen-regulated manner. TRPV1 is upregulated in peritoneal and ovarian endometriosis patients, but there is no information about TRPA1 and their pathophysiological significances. In this study, patients undergoing laparoscopic surgery were investigated: severe dysmenorrhoea due to rectosigmoid deep infiltrating endometriosis ( n = 15), uterine fibroid-induced moderate dysmenorrhoea ( n = 7) and tubal infertility with no pain ( n = 6). TRPA1 and TRPV1 mRNA and protein expressions were determined by quantitative polymerase chain reaction and semi-quantitative immunohistochemistry from the endometrium samples taken by curettage. Results were correlated with the clinical characteristics including pain intensity. TRPA1 and TRPV1 receptors were expressed in the healthy human endometrium at mRNA and protein levels. Sparse, scattered cytoplasmic TRPA1 and TRPV1 immunopositivities were found in the stroma and epithelial layers. We detected upregulated mRNA levels in deep infiltrating endometriosis lesions, and TRPV1 gene expression was also elevated in autocontrol endometrium of deep infiltrating endometriosis patients. Histological scoring revealed significant TRPA1 and TRPV1 difference between deep infiltrating endometriosis stroma and epithelium, and in deep infiltrating endometriosis epithelium compared to control samples. Besides, we measured elevated stromal TRPV1 immunopositivity in deep infiltrating endometriosis. Stromal TRPA1 and TRPV1 immunoreactivities strongly correlated with dysmenorrhoea severity, as well TRPV1 expression on ectopic epithelial cells and macrophages with dyspareunia. Epithelial TRPA1 and stromal TRPV1 immunopositivity also positively correlated with dyschezia severity. We provide the first evidence for the presence of non-neuronal TRPA1 receptor in the healthy human endometrium and confirm the expression of TRPV1 channels. Their upregulations in rectosigmoid deep infiltrating endometriosis lesions and correlations with pain intensity suggest potential roles in pathophysiological mechanisms of the disease.

Transient global amnesia: implicit/explicit memory dissociation and PET assessment of brain perfusion and oxygen metabolism in the acute stage.

PubMed

Eustache, F; Desgranges, B; Petit-Taboué, M C; de la Sayette, V; Piot, V; Sablé, C; Marchal, G; Baron, J C

1997-09-01

To assess explicit memory and two components of implicit memory--that is, perceptual-verbal skill learning and lexical-semantic priming effects--as well as resting cerebral blood flow (CBF) and oxygen metabolism (CMRO2) during the acute phase of transient global amnesia. In a 59 year old woman, whose amnestic episode fulfilled all current criteria for transient global amnesia, a neuropsychological protocol was administered, including word learning, story recall, categorical fluency, mirror reading, and word stem completion tasks. PET was performed using the (15)O steady state inhalation method, while the patient still exhibited severe anterograde amnesia and was interleaved with the cognitive tests. There was a clear cut dissociation between impaired long term episodic memory and preserved implicit memory for its two components. Categorical fluency was significantly altered, suggesting word retrieval strategy--rather than semantic memory--impairment. The PET study disclosed a reduced CMRO2 with relatively or fully preserved CBF in the left prefrontotemporal cortex and lentiform nucleus, and the reverse pattern over the left occipital cortex. The PET alterations with patchy CBF-CMRO2 uncoupling would be compatible with a migraine-like phenomenon and indicate that the isolated assessment of perfusion in transient global amnesia may be misleading. The pattern of metabolic depression, with sparing of the hippocampal area, is one among the distinct patterns of brain dysfunction that underlie the (apparently) uniform clinical presentation of transient global amnesia. The finding of a left prefrontal hypometabolism in the face of impaired episodic memory and altered verbal fluency would fit present day concepts from PET activation studies about the role of this area in episodic and semantic memory encoding/retrieval. Likewise, the changes affecting the lenticular nucleus but sparing the caudate would be consistent with the normal performance in perceptual-verbal skill learning. Finally, unaltered lexical-semantic priming effects, despite left temporal cortex hypometabolism, suggest that these processes are subserved by a more distributed neocortical network.

Developmental evolution of flowering plant pollen tube cell walls: callose synthase (CalS) gene expression patterns

PubMed Central

2011-01-01

Background A number of innovations underlie the origin of rapid reproductive cycles in angiosperms. A critical early step involved the modification of an ancestrally short and slow-growing pollen tube for faster and longer distance transport of sperm to egg. Associated with this shift are the predominantly callose (1,3-β-glucan) walls and septae (callose plugs) of angiosperm pollen tubes. Callose synthesis is mediated by callose synthase (CalS). Of 12 CalS gene family members in Arabidopsis, only one (CalS5) has been directly linked to pollen tube callose. CalS5 orthologues are present in several monocot and eudicot genomes, but little is known about the evolutionary origin of CalS5 or what its ancestral function may have been. Results We investigated expression of CalS in pollen and pollen tubes of selected non-flowering seed plants (gymnosperms) and angiosperms within lineages that diverged below the monocot/eudicot node. First, we determined the nearly full length coding sequence of a CalS5 orthologue from Cabomba caroliniana (CcCalS5) (Nymphaeales). Semi-quantitative RT-PCR demonstrated low CcCalS5 expression within several vegetative tissues, but strong expression in mature pollen. CalS transcripts were detected in pollen tubes of several species within Nymphaeales and Austrobaileyales, and comparative analyses with a phylogenetically diverse group of sequenced genomes indicated homology to CalS5. We also report in silico evidence of a putative CalS5 orthologue from Amborella. Among gymnosperms, CalS5 transcripts were recovered from germinating pollen of Gnetum and Ginkgo, but a novel CalS paralog was instead amplified from germinating pollen of Pinus taeda. Conclusion The finding that CalS5 is the predominant callose synthase in pollen tubes of both early-diverging and model system angiosperms is an indicator of the homology of their novel callosic pollen tube walls and callose plugs. The data suggest that CalS5 had transient expression and pollen-specific functions in early seed plants and was then recruited to novel expression patterns and functions within pollen tube walls in an ancestor of extant angiosperms. PMID:21722365

Switching on Flowers: Transient LEAFY Induction Reveals Novel Aspects of the Regulation of Reproductive Development in Arabidopsis

PubMed Central

Wagner, Doris; Meyerowitz, Elliot M.

2011-01-01

Developmental fate decisions in cell populations fundamentally depend on at least two parameters: a signal that is perceived by the cell and the intrinsic ability of the cell to respond to the signal. The same regulatory logic holds for phase transitions in the life cycle of an organism, for example the switch to reproductive development in flowering plants. Here we have tested the response of the monocarpic plant species Arabidopsis thaliana to a signal that directs flower formation, the plant-specific transcription factor LEAFY (LFY). Using transient steroid-dependent LEAFY (LFY) activation in lfy null mutant Arabidopsis plants, we show that the plant’s competence to respond to the LFY signal changes during development. Very early in the life cycle, the plant is not competent to respond to the signal. Subsequently, transient LFY activation can direct primordia at the flanks of the shoot apical meristem to adopt a floral fate. Finally, the plants acquire competence to initiate the flower-patterning program in response to transient LFY activation. Similar to a perennial life strategy, we did not observe reprogramming of all primordia after perception of the transient signal, instead only a small number of meristems responded, followed by reversion to the prior developmental program. The ability to initiate flower formation and to direct flower patterning in response to transient LFY upregulation was dependent on the known direct LFY target APETALA1 (AP1). Prolonged LFY or activation could alter the developmental gradient and bypass the requirement for AP1. Prolonged high AP1 levels, in turn, can also alter the plants’ competence. Our findings shed light on how plants can fine-tune important phase transitions and developmental responses. PMID:22639600

Modeling the Regulatory Mechanisms by Which NLRX1 Modulates Innate Immune Responses to Helicobacter pylori Infection

PubMed Central

Philipson, Casandra W.; Bassaganya-Riera, Josep; Viladomiu, Monica; Kronsteiner, Barbara; Abedi, Vida; Hoops, Stefan; Michalak, Pawel; Kang, Lin; Girardin, Stephen E.; Hontecillas, Raquel

2015-01-01

Helicobacter pylori colonizes half of the world’s population as the dominant member of the gastric microbiota resulting in a lifelong chronic infection. Host responses toward the bacterium can result in asymptomatic, pathogenic or even favorable health outcomes; however, mechanisms underlying the dual role of H. pylori as a commensal versus pathogenic organism are not well characterized. Recent evidence suggests mononuclear phagocytes are largely involved in shaping dominant immunity during infection mediating the balance between host tolerance and succumbing to overt disease. We combined computational modeling, bioinformatics and experimental validation in order to investigate interactions between macrophages and intracellular H. pylori. Global transcriptomic analysis on bone marrow-derived macrophages (BMDM) in a gentamycin protection assay at six time points unveiled the presence of three sequential host response waves: an early transient regulatory gene module followed by sustained and late effector responses. Kinetic behaviors of pattern recognition receptors (PRRs) are linked to differential expression of spatiotemporal response waves and function to induce effector immunity through extracellular and intracellular detection of H. pylori. We report that bacterial interaction with the host intracellular environment caused significant suppression of regulatory NLRC3 and NLRX1 in a pattern inverse to early regulatory responses. To further delineate complex immune responses and pathway crosstalk between effector and regulatory PRRs, we built a computational model calibrated using time-series RNAseq data. Our validated computational hypotheses are that: 1) NLRX1 expression regulates bacterial burden in macrophages; and 2) early host response cytokines down-regulate NLRX1 expression through a negative feedback circuit. This paper applies modeling approaches to characterize the regulatory role of NLRX1 in mechanisms of host tolerance employed by macrophages to respond to and/or to co-exist with intracellular H. pylori. PMID:26367386

Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins

PubMed Central

Beissert, Tim; Koste, Lars; Perkovic, Mario; Walzer, Kerstin C.; Erbar, Stephanie; Selmi, Abderraouf; Diken, Mustafa; Kreiter, Sebastian; Türeci, Özlem; Sahin, Ugur

2017-01-01

Among nucleic acid–based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdown. To reduce pattern recognition receptor stimulation and unleash suppressed saRNA translation, this study co-delivered non-replicating mRNA encoding vaccinia virus immune evasion proteins E3, K3, and B18. It was shown that E3 is far superior to K3 or B18 as a highly potent blocker of PKR activation and of interferon (IFN)-β upregulation. B18, in contrast, is superior in controlling OAS1, a key IFN-inducible gene involved in viral RNA degradation. By combining all three vaccinia proteins, the study achieved significant suppression of PKR and IFN pathway activation in vitro and enhanced expression of saRNA-encoded genes of interest both in vitro and in vivo. This approach promises to overcome key hurdles of saRNA gene delivery. Its application may improve the bioavailability of the encoded protein, and reduce the effective dose and correspondingly the cost of goods of manufacture in the various fields where saRNA utilization is envisioned. PMID:28877647

Prolonged administration of a dithiol antioxidant protects against ventricular remodeling due to ischemia-reperfusion in mice.

PubMed

Ambler, S Kelly; Hodges, Yvonne K; Jones, Gayle M; Long, Carlin S; Horwitz, Lawrence D

2008-09-01

The prolonged production of reactive oxygen species due to ischemia-reperfusion (I/R) is a potential cause of the pathological remodeling that frequently precedes heart failure. We tested the ability of a potent dithiol antioxidant, bucillamine, to protect against the long-term consequences of I/R injury in a murine model of myocardial infarction. After transiently occluding the left anterior descending coronary artery for 30 min, saline or bucillamine (10 microg/g body wt) was injected intravenously as a bolus within the first 5 min of reperfusion. The antioxidant treatment continued with daily subcutaneous injections for 4 wk. There were no differences in infarct sizes between bucillamine- and saline-treated animals. After 4 wk of reperfusion, cardiac hypertrophy was decreased by bucillamine treatment (ventricular weight-to-body weight ratios: I/R + saline, 4.5 +/- 0.2 mg/g vs. I/R + bucillamine, 4.2 +/- 0.1 mg/g; means +/- SE; P < 0.05). Additionally, the hearts of bucillamine-treated mice had improved contractile function (echocardiographic measurement of fractional shortening) relative to saline controls: I/R + saline, 32 +/- 3%, versus I/R + bucillamine, 41 +/- 4% (P < 0.05). Finally, I/R-induced injury in the saline-treated mice was accompanied by a fetal pattern of gene expression determined by ribonuclease protection assay that was consistent with pathological cardiac hypertrophy and remodeling [increased atrial natriuretic peptide, beta-myosin heavy chain (MHC), skeletal alpha-actin; decreased sarco(endo)plasmic reticulum Ca2+ ATPase 2a, and alpha-MHC-to-beta-MHC ratio]. These changes in gene expression were significantly attenuated by bucillamine. Therefore, treatment with a dithiol antioxidant for 4 wk after I/R preserved ventricular function and prevented the abnormal pattern of gene expression associated with pathological cardiac remodeling.

Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins.

PubMed

Beissert, Tim; Koste, Lars; Perkovic, Mario; Walzer, Kerstin C; Erbar, Stephanie; Selmi, Abderraouf; Diken, Mustafa; Kreiter, Sebastian; Türeci, Özlem; Sahin, Ugur

2017-12-01

Among nucleic acid-based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdown. To reduce pattern recognition receptor stimulation and unleash suppressed saRNA translation, this study co-delivered non-replicating mRNA encoding vaccinia virus immune evasion proteins E3, K3, and B18. It was shown that E3 is far superior to K3 or B18 as a highly potent blocker of PKR activation and of interferon (IFN)-β upregulation. B18, in contrast, is superior in controlling OAS1, a key IFN-inducible gene involved in viral RNA degradation. By combining all three vaccinia proteins, the study achieved significant suppression of PKR and IFN pathway activation in vitro and enhanced expression of saRNA-encoded genes of interest both in vitro and in vivo. This approach promises to overcome key hurdles of saRNA gene delivery. Its application may improve the bioavailability of the encoded protein, and reduce the effective dose and correspondingly the cost of goods of manufacture in the various fields where saRNA utilization is envisioned.

Dormancy-associated MADS-box genes and microRNAs jointly control dormancy transition in pear (Pyrus pyrifolia white pear group) flower bud

PubMed Central

Niu, Qingfeng; Li, Jianzhao; Cai, Danying; Qian, Minjie; Jia, Huimin; Bai, Songling; Hussain, Sayed; Liu, Guoqin; Teng, Yuanwen; Zheng, Xiaoyan

2016-01-01

Bud dormancy in perennial plants is indispensable to survival over winter and to regrowth and development in the following year. However, the molecular pathways of endo-dormancy induction, maintenance, and release are still unclear, especially in fruit crops. To identify genes with roles in regulating endo-dormancy, 30 MIKCC-type MADS-box genes were identified in the pear genome and characterized. The 30 genes were analysed to determine their phylogenetic relationships with homologous genes, genome locations, gene structure, tissue-specific transcript profiles, and transcriptional patterns during flower bud dormancy in ‘Suli’ pear (Pyrus pyrifolia white pear group). The roles in regulating bud dormancy varied among the MIKC gene family members. Yeast one-hybrid and transient assays showed that PpCBF enhanced PpDAM1 and PpDAM3 transcriptional activity during the induction of dormancy, probably by binding to the C-repeat/DRE binding site, while DAM proteins inhibited the transcriptional activity of PpFT2 during dormancy release. In the small RNA-seq analysis, 185 conserved, 24 less-conserved, and 32 pear-specific miRNAs with distinct expression patterns during bud dormancy were identified. Joint analyses of miRNAs and MIKC genes together with degradome data showed that miR6390 targeted PpDAM transcripts and degraded them to release PpFT2. Our data show that cross-talk among PpCBF, PpDAM, PpFT2, and miR6390 played important roles in regulating endo-dormancy. A model for the molecular mechanism of dormancy transition is proposed: short-term chilling in autumn activates the accumulation of CBF, which directly promotes DAM expression; DAM subsequently inhibits FT expression to induce endo-dormancy, and miR6390 degrades DAM genes to release endo-dormancy. PMID:26466664

A SHATTERPROOF-like gene controls ripening in non-climacteric strawberries, and auxin and abscisic acid antagonistically affect its expression.

PubMed

Daminato, Margherita; Guzzo, Flavia; Casadoro, Giorgio

2013-09-01

Strawberries (Fragaria×ananassa) are false fruits the ripening of which follows the non-climacteric pathway. The role played by a C-type MADS-box gene [SHATTERPROOF-like (FaSHP)] in the ripening of strawberries has been studied by transiently modifying gene expression through either over-expression or RNA-interference-mediated down-regulation. The altered expression of the FaSHP gene caused a change in the time taken by the over-expressing and the down- regulated fruits to attain the pink stage, which was slightly shorter and much longer, respectively, compared to controls. In parallel with the modified ripening times, the metabolome components and the expression of ripening-related genes also appeared different in the transiently modified fruits. Differences in the response time of the analysed genes suggest that FaSHP can control the expression of ripening genes either directly or indirectly through other transcription factor-encoding genes. Because fleshy strawberries are false fruits these results indicate that C-type MADS-box genes like SHATTERPROOF may act as modulators of ripening in fleshy fruit-like structures independently of their anatomical origin. Treatment of strawberries with either auxin or abscisic acid had antagonistic impacts on both the expression of FaSHP and the expression of ripening-related genes and metabolome components.

Longitudinal Studies of a B Cell Derived Signature of Tolerance in Renal Transplant Recipients

PubMed Central

Newell, Kenneth A.; Asare, Adam; Sanz, Ignacio; Wei, Chungwen; Rosenberg, Alexander; Gao, Zhong; Kanaparthi, Sai; Asare, Smita; Lim, Noha; Stahly, Michael; Howell, Michael; Knechtle, Stuart; Kirk, Allan; Marks, William H.; Kawai, Tatsuo; Spitzer, Thomas; Tolkoff-Rubin, Nina; Sykes, Megan; Sachs, David H.; Cosimi, A. Benedict; Burlingham, William J.; Phippard, Deborah; Turka, Laurence A.

2016-01-01

Biomarkers of transplant tolerance would enhance the safety and feasibility of clinical tolerance trials and potentially facilitate management of patients receiving immunosuppression. To this end, we examined blood from spontaneously tolerant renal transplant recipients and patients enrolled in two interventional tolerance trials using flow cytometry and gene expression profiling. Using a previously reported tolerant cohort as well as newly identified tolerant patients we confirmed our previous finding that tolerance was associated with increased expression of B cell-associated genes relative to immunosuppressed patients. This was not accounted for merely by an increase in total B cell numbers, but was associated with the increased frequencies of transitional and naïve B cells. Moreover, serial measurements of gene expression demonstrated that this pattern persisted over several years although patients receiving immunosuppression also displayed an increase in the two most dominant tolerance-related B cell genes, IGKV1D-13 and IGLL-1, over time. Importantly, patients rendered tolerant via induction of transient mixed chimerism, and those weaned to minimal immunosuppression, showed similar increases in IGKV1D-13 as did spontaneously tolerant individuals. Collectively, these findings support the notion that alterations in B cells may be a common theme for tolerant kidney transplant recipients, and a useful monitoring tool in prospective trials. PMID:26461968

The activity of spontaneous action potentials in developing hair cells is regulated by Ca(2+)-dependence of a transient K+ current.

PubMed

Levic, Snezana; Lv, Ping; Yamoah, Ebenezer N

2011-01-01

Spontaneous action potentials have been described in developing sensory systems. These rhythmic activities may have instructional roles for the functional development of synaptic connections. The importance of spontaneous action potentials in the developing auditory system is underpinned by the stark correlation between the time of auditory system functional maturity, and the cessation of spontaneous action potentials. A prominent K(+) current that regulates patterning of action potentials is I(A). This current undergoes marked changes in expression during chicken hair cell development. Although the properties of I(A) are not normally classified as Ca(2+)-dependent, we demonstrate that throughout the development of chicken hair cells, I(A) is greatly reduced by acute alterations of intracellular Ca(2+). As determinants of spike timing and firing frequency, intracellular Ca(2+) buffers shift the activation and inactivation properties of the current to more positive potentials. Our findings provide evidence to demonstrate that the kinetics and functional expression of I(A) are tightly regulated by intracellular Ca(2+). Such feedback mechanism between the functional expression of I(A) and intracellular Ca(2+) may shape the activity of spontaneous action potentials, thus potentially sculpting synaptic connections in an activity-dependent manner in the developing cochlea. © 2011 Levic et al.

Isolation and functional characterization of a τ-cadinol synthase, a new sesquiterpene synthase from Lavandula angustifolia.

PubMed

Jullien, Frédéric; Moja, Sandrine; Bony, Aurélie; Legrand, Sylvain; Petit, Cécile; Benabdelkader, Tarek; Poirot, Kévin; Fiorucci, Sébastien; Guitton, Yann; Nicolè, Florence; Baudino, Sylvie; Magnard, Jean-Louis

2014-01-01

In this paper we characterize three sTPSs: a germacrene D (LaGERDS), a (E)-β-caryophyllene (LaCARS) and a τ-cadinol synthase (LaCADS). τ-cadinol synthase is reported here for the first time and its activity was studied in several biological models including transiently or stably transformed tobacco species. Three dimensional structure models of LaCADS and Ocimum basilicum γ-cadinene synthase were built by homology modeling using the template structure of Gossypium arboreum δ-cadinene synthase. The depiction of their active site organization provides evidence of the global influence of the enzymes on the formation of τ-cadinol: instead of a unique amino-acid, the electrostatic properties and solvent accessibility of the whole active site in LaCADS may explain the stabilization of the cadinyl cation intermediate. Quantitative PCR performed from leaves and inflorescences showed two patterns of expression. LaGERDS and LaCARS were mainly expressed during early stages of flower development and, at these stages, transcript levels paralleled the accumulation of the corresponding terpene products (germacrene D and (E)-β-caryophyllene). By contrast, the expression level of LaCADS was constant in leaves and flowers. Phylogenetic analysis provided informative results on potential duplication process leading to sTPS diversification in lavender.

Analysis of a Plant Transcriptional Regulatory Network Using Transient Expression Systems.

PubMed

Díaz-Triviño, Sara; Long, Yuchen; Scheres, Ben; Blilou, Ikram

2017-01-01

In plant biology, transient expression systems have become valuable approaches used routinely to rapidly study protein expression, subcellular localization, protein-protein interactions, and transcriptional activity prior to in vivo studies. When studying transcriptional regulation, luciferase reporter assays offer a sensitive readout for assaying promoter behavior in response to different regulators or environmental contexts and to confirm and assess the functional relevance of predicted binding sites in target promoters. This chapter aims to provide detailed methods for using luciferase reporter system as a rapid, efficient, and versatile assay to analyze transcriptional regulation of target genes by transcriptional regulators. We describe a series of optimized transient expression systems consisting of Arabidopsis thaliana protoplasts, infiltrated Nicotiana benthamiana leaves, and human HeLa cells to study the transcriptional regulations of two well-characterized transcriptional regulators SCARECROW (SCR) and SHORT-ROOT (SHR) on one of their targets, CYCLIN D6 (CYCD6).Here, we illustrate similarities and differences in outcomes when using different systems. The plant-based systems revealed that the SCR-SHR complex enhances CYCD6 transcription, while analysis in HeLa cells showed that the complex is not sufficient to strongly induce CYCD6 transcription, suggesting that additional, plant-specific regulators are required for full activation. These results highlight the importance of the system and suggest that including heterologous systems, such as HeLa cells, can provide a more comprehensive analysis of a complex gene regulatory network.

Effects of heat stress and starvation on clonal odontoblast-like cells.

PubMed

Morotomi, Takahiko; Kitamura, Chiaki; Toyono, Takashi; Okinaga, Toshinori; Washio, Ayako; Saito, Noriko; Nishihara, Tatsuji; Terashita, Masamichi; Anan, Hisashi

2011-07-01

Heat stress during restorative procedures, particularly under severe starvation conditions, can trigger damage to dental pulp. In the present study, we examined effects of heat stress on odontoblastic activity and inflammatory responses in an odontoblast-like cell line (KN-3) under serum-starved conditions. Viability, nuclear structures, and inflammatory responses of KN-3 cells were examined in culture medium containing 10% or 1% serum after exposure to heat stress at 43°C for 45 minutes. Gene expression of extracellular matrices, alkaline phosphatase activity, and detection of extracellular calcium deposition in cells exposed to heat stress were also examined. Reduced viability and apoptosis were transiently induced in KN-3 cells during the initial phases after heat stress; thereafter, cells recovered their viability. The cytotoxic effects of heat stress were enhanced under serum-starved conditions. Heat stress also strongly up-regulated expression of heat shock protein 25 as well as transient expression of tumor necrosis factor-alpha, interleukin-6, and cyclooxygenase-2 in KN-3 cells. In contrast, expression of type-1 collagen, runt-related transcription factor 2, and dentin sialophosphoprotein were not inhibited by heat stress although starvation suppressed ALP activity and delayed progression of calcification. Odontoblast-like cells showed thermoresistance with transient inflammatory responses and without loss of calcification activity, and their thermoresistance and calcification activity were influenced by nutritional status. Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Directly transforming PCR-amplified DNA fragments into plant cells is a versatile system that facilitates the transient expression assay.

PubMed

Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan

2013-01-01

A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells.

Directly Transforming PCR-Amplified DNA Fragments into Plant Cells Is a Versatile System That Facilitates the Transient Expression Assay

PubMed Central

Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan

2013-01-01

A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells. PMID:23468926

Chronology of Islet Differentiation Revealed By Temporal Cell Labeling

PubMed Central

Miyatsuka, Takeshi; Li, Zhongmei; German, Michael S.

2009-01-01

OBJECTIVE Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene. RESEARCH DESIGN AND METHODS In order to separate the transient neurogenin 3 –expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus. RESULTS In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas. CONCLUSIONS The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells. PMID:19478145

Transient Dynamics Simulation of Airflow in a CT-Scanned Human Airway Tree: More or Fewer Terminal Bronchi?

PubMed Central

Zhang, Baihua; Li, Jianhua; Yue, Yong; Qian, Wei

2017-01-01

Using computational fluid dynamics (CFD) method, the feasibility of simulating transient airflow in a CT-based airway tree with more than 100 outlets for a whole respiratory period is studied, and the influence of truncations of terminal bronchi on CFD characteristics is investigated. After an airway model with 122 outlets is extracted from CT images, the transient airflow is simulated. Spatial and temporal variations of flow velocity, wall pressure, and wall shear stress are presented; the flow pattern and lobar distribution of air are gotten as well. All results are compared with those of a truncated model with 22 outlets. It is found that the flow pattern shows lobar heterogeneity that the near-wall air in the trachea is inhaled into the upper lobe while the center flow enters the other lobes, and the lobar distribution of air is significantly correlated with the outlet area ratio. The truncation decreases airflow to right and left upper lobes and increases the deviation of airflow distributions between inspiration and expiration. Simulating the transient airflow in an airway tree model with 122 bronchi using CFD is feasible. The model with more terminal bronchi decreases the difference between the lobar distributions at inspiration and at expiration. PMID:29333194

Associations of different phenotypes of wheezing illness in early childhood with environmental variables implicated in the aetiology of asthma.

PubMed

Granell, Raquel; Sterne, Jonathan A C; Henderson, John

2012-01-01

Asthma is a complex heterogeneous disease that has increased in prevalence in many industrialised countries. However, the causes of asthma inception remain elusive. Consideration of sub-phenotypes of wheezing may reveal important clues to aetiological risk factors. Longitudinal phenotypes capturing population heterogeneity in wheezing reports from birth to 7 years were derived using latent class analysis in the Avon Longitudinal Study of Parents and Children (ALSPAC). Probability of class membership was used to examine the association between five wheezing phenotypes (transient early, prolonged early, intermediate-onset, late-onset, persistent) and early life risk factors for asthma. Phenotypes had similar patterns and strengths of associations with early environmental factors. Comparing transient early with prolonged early wheezing showed a similar pattern of association with most exposure variables considered in terms of the direction of the effect estimates but with prolonged early wheezing tending to have stronger associations than transient early wheezing except for parity and day care attendance. Associations with early life risk factors suggested that prolonged early wheeze might be a severe form of transient early wheezing. Although differences were found in the associations of early life risk factors with individual phenotypes, these did not point to novel aetiological pathways. Persistent wheezing phenotype has features suggesting overlap of early and late-onset phenotypes.

Natural convection heat transfer in an oscillating vertical cylinder

PubMed Central

Ali Shah, Nehad; Tassaddiq, Asifa; Mustapha, Norzieha; Kechil, Seripah Awang

2018-01-01

This paper studies the heat transfer analysis caused due to free convection in a vertically oscillating cylinder. Exact solutions are determined by applying the Laplace and finite Hankel transforms. Expressions for temperature distribution and velocity field corresponding to cosine and sine oscillations are obtained. The solutions that have been obtained for velocity are presented in the forms of transient and post-transient solutions. Moreover, these solutions satisfy both the governing differential equation and all imposed initial and boundary conditions. Numerical computations and graphical illustrations are used in order to study the effects of Prandtl and Grashof numbers on velocity and temperature for various times. The transient solutions for both cosine and sine oscillations are also computed in tables. It is found that, the transient solutions are of considerable interest up to the times t = 15 for cosine oscillations and t = 1.75 for sine oscillations. After these moments, the transient solutions can be neglected and, the fluid moves according with the post-transient solutions. PMID:29304161

Natural convection heat transfer in an oscillating vertical cylinder.

PubMed

Khan, Ilyas; Ali Shah, Nehad; Tassaddiq, Asifa; Mustapha, Norzieha; Kechil, Seripah Awang

2018-01-01

This paper studies the heat transfer analysis caused due to free convection in a vertically oscillating cylinder. Exact solutions are determined by applying the Laplace and finite Hankel transforms. Expressions for temperature distribution and velocity field corresponding to cosine and sine oscillations are obtained. The solutions that have been obtained for velocity are presented in the forms of transient and post-transient solutions. Moreover, these solutions satisfy both the governing differential equation and all imposed initial and boundary conditions. Numerical computations and graphical illustrations are used in order to study the effects of Prandtl and Grashof numbers on velocity and temperature for various times. The transient solutions for both cosine and sine oscillations are also computed in tables. It is found that, the transient solutions are of considerable interest up to the times t = 15 for cosine oscillations and t = 1.75 for sine oscillations. After these moments, the transient solutions can be neglected and, the fluid moves according with the post-transient solutions.

Sensitivity analysis of reactive ecological dynamics.

PubMed

Verdy, Ariane; Caswell, Hal

2008-08-01

Ecological systems with asymptotically stable equilibria may exhibit significant transient dynamics following perturbations. In some cases, these transient dynamics include the possibility of excursions away from the equilibrium before the eventual return; systems that exhibit such amplification of perturbations are called reactive. Reactivity is a common property of ecological systems, and the amplification can be large and long-lasting. The transient response of a reactive ecosystem depends on the parameters of the underlying model. To investigate this dependence, we develop sensitivity analyses for indices of transient dynamics (reactivity, the amplification envelope, and the optimal perturbation) in both continuous- and discrete-time models written in matrix form. The sensitivity calculations require expressions, some of them new, for the derivatives of equilibria, eigenvalues, singular values, and singular vectors, obtained using matrix calculus. Sensitivity analysis provides a quantitative framework for investigating the mechanisms leading to transient growth. We apply the methodology to a predator-prey model and a size-structured food web model. The results suggest predator-driven and prey-driven mechanisms for transient amplification resulting from multispecies interactions.

Analytical solution of the transient temperature profile in gain medium of passively Q-switched microchip laser.

PubMed

Han, Xiahui; Li, Jianlang

2014-11-01

The transient temperature evolution in the gain medium of a continuous wave (CW) end-pumped passively Q-switched microchip (PQSM) laser is analyzed. By approximating the time-dependent population inversion density as a sawtooth function of time and treating the time-dependent pump absorption of a CW end-pumped PQSM laser as the superposition of an infinite series of short pumping pulses, the analytical expressions of transient temperature evolution and distribution in the gain medium for four- and three-level laser systems, respectively, are given. These analytical solutions are applied to evaluate the transient temperature evolution and distribution in the gain medium of CW end-pumped PQSM Nd:YAG and Yb:YAG lasers.

Transient expression and activity of human DNA polymerase iota in loach embryos.

PubMed

Makarova, Irina V; Kazakov, Andrey A; Makarova, Alena V; Khaidarova, Nella V; Kozikova, Larisa V; Nenasheva, Valentina V; Gening, Leonid V; Tarantul, Vyacheslav Z; Andreeva, Ludmila E

2012-02-01

Human DNA polymerase iota (Pol ι) is a Y-family DNA polymerase with unusual biochemical properties and not fully understood functions. Pol ι preferentially incorporates dGTP opposite template thymine. This property can be used to monitor Pol ι activity in the presence of other DNA polymerases, e.g. in cell extracts of tissues and tumors. We have now confirmed the specificity and sensitivity of the method of Pol ι activity detection in cell extracts using an animal model of loach Misgurnus fossilis embryos transiently expressing human Pol ι. The overexpression of Pol ι was shown to be accompanied by an increase in abnormalities in development and the frequency of pycnotic nuclei in fish embryos. Further analysis of fish embryos with constitutive or regulated Pol ι expression may provide insights into Pol ι functions in vertebrate animals.

Long-term spironolactone treatment reduces coronary TRPC expression, vasoconstriction, and atherosclerosis in metabolic syndrome pigs.

PubMed

Li, Wennan; Chen, Xingjuan; Riley, Ashley M; Hiett, S Christopher; Temm, Constance J; Beli, Eleni; Long, Xin; Chakraborty, Saikat; Alloosh, Mouhamad; White, Fletcher A; Grant, Maria B; Sturek, Michael; Obukhov, Alexander G

2017-09-01

Coronary transient receptor potential canonical (TRPC) channel expression is elevated in metabolic syndrome (MetS). However, differential contribution of TRPCs to coronary pathology in MetS is not fully elucidated. We investigated the roles of TRPC1 and TRPC6 isoforms in coronary arteries of MetS pigs and determined whether long-term treatment with a mineralocorticoid receptor inhibitor, spironolactone, attenuates coronary TRPC expression and associated dysfunctions. MetS coronary arteries exhibited significant atherosclerosis, endothelial dysfunction, and increased histamine-induced contractions. Immunohistochemical studies revealed that TRPC6 immunostaining was significantly greater in the medial layer of MetS pig coronary arteries compared to that in Lean pigs, whereas little TRPC6 immunostaining was found in atheromas. Conversely, TRPC1 immunostaining was weak in the medial layer but strong in MetS atheromas, where it was predominantly localized to macrophages. Spironolactone treatment significantly decreased coronary TRPC expression and dysfunctions in MetS pigs. In vivo targeted delivery of the dominant-negative (DN)-TRPC6 cDNA to the coronary wall reduced histamine-induced calcium transients in the MetS coronary artery medial layer, implying a role for TRPC6 in mediating calcium influx in MetS coronary smooth muscles. Monocyte adhesion was increased in Lean pig coronary arteries cultured in the presence of aldosterone; and spironolactone antagonized this effect, suggesting that coronary mineralocorticoid receptor activation may regulate macrophage infiltration. TRPC1 expression in atheroma macrophages was associated with advanced atherosclerosis, whereas medial TRPC6 upregulation correlated with increased histamine-induced calcium transients and coronary contractility. We propose that long-term spironolactone treatment may be a therapeutic strategy to decrease TRPC expression and coronary pathology associated with MetS.

Identification and Transcript Analysis of the TCP Transcription Factors in the Diploid Woodland Strawberry Fragaria vesca

PubMed Central

Wei, Wei; Hu, Yang; Cui, Meng-Yuan; Han, Yong-Tao; Gao, Kuan; Feng, Jia-Yue

2016-01-01

Plant-specific TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTORS (TCP) transcription factors play versatile functions in multiple processes of plant growth and development. However, no systematic study has been performed in strawberry. In this study, 19 FvTCP genes were identified in the diploid woodland strawberry (Fragaria vesca) accession Heilongjiang-3. Phylogenetic analysis suggested that the FvTCP genes were classified into two main classes, with the second class further divided into two subclasses, which was supported by the exon-intron organizations and the conserved motif structures. Promoter analysis revealed various cis-acting elements related to growth and development, hormone and/or stress responses. We analyzed FvTCP gene transcript accumulation patterns in different tissues and fruit developmental stages. Among them, 12 FvTCP genes exhibited distinct tissue-specific transcript accumulation patterns. Eleven FvTCP genes were down-regulated in different fruit developmental stages, while five FvTCP genes were up-regulated. Transcripts of FvTCP genes also varied with different subcultural propagation periods and were induced by hormone treatments and biotic and abiotic stresses. Subcellular localization analysis showed that six FvTCP-GFP fusion proteins showed distinct localizations in Arabidopsis mesophyll protoplasts. Notably, transient over-expression of FvTCP9 in strawberry fruits dramatically affected the expression of a series of genes implicated in fruit development and ripening. Taken together, the present study may provide the basis for functional studies to reveal the role of this gene family in strawberry growth and development. PMID:28066489

Production of the Main Celiac Disease Autoantigen by Transient Expression in Nicotiana benthamiana

PubMed Central

Marín Viegas, Vanesa S.; Acevedo, Gonzalo R.; Bayardo, Mariela P.; Chirdo, Fernando G.; Petruccelli, Silvana

2015-01-01

Celiac Disease (CD) is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2), is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion) and vacuolar (C-terminal KISIA fusion) TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9- to 16-fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP) construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB) were induced by the ELP, with a consequent two-fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant-produced TG2 to develop screening assays. In conclusion, the combination of subcellular sorting strategy with co-expression with a PB inducing construct was sufficient to increase TG2 protein yields. This type of approach could be extended to other problematic proteins, highlighting the advantages of plant based production platforms. PMID:26648956

System Re-set: High LET Radiation or Transient Musculoskeletal Disuse Cause Lasting Changes in Oxidative Defense Pathways Within Bone

NASA Technical Reports Server (NTRS)

Kumar, Akhilesh; Chatterjee, A.; Alwood, Joshua S.; Dvorochkin, Natalya; Almeida, Eduardo A. C.

2011-01-01

Six months post-IR, there were no notable changes in skeletal expression of 84 principal genes in the p53 signaling pathway due to low dose IR (0.5Gy), HU, or both. In contrast, numerous genes relevant to oxidative stress were regulated by the treatments, typically in a direction indicative of increased oxidative stress and impaired defense. IR and HU independently reduced (between 0.46 to 0.88 fold) expression levels of Noxa1, Gpx3, Prdx2, Prdx3, and Zmynd17. Surprisingly, transient HU alone (sham-irradiated) decreased expression of several redox-related genes (Gpx1,Gstk1, Prdx1, Txnrd2), which were not affected significantly by IR alone. Irradiation increased (1.13 fold) expression of a gene responsible for production of superoxides by neutrophils (NCF2). Of interest, only combined treatment with HU and IR led to increased expression levels of Ercc2, (1.19 fold), a DNA excision repair enzyme. Differences in gene expression levels may reflect a change in gene expression on a per cell basis, a shift in the repertoire of specific cell types within the tissue, or both. Serum nitrite/nitrate levels were elevated to comparable levels (1.6-fold) due to IR, HU or both, indicative of elevated systemic nitrosyl stress. CONCLUSIONS The magnitude of changes in skeletal expression of oxidative stress-related genes six months after irradiation and/or transient unloading tended to be relatively modest (0.46-1.15 fold), whereas the p53 pathway was not affected. The finding that many different oxidative stress-related genes differed from controls at this late time point implicates a generalized impairment of oxidative defense within skeletal tissue, which coincides with both profound radiation damage to osteoprogenitors/stem cells in bone marrow and impaired remodeling of mineralized tissue.

Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium.

PubMed

Shabir, Saqib; Cross, William; Kirkwood, Lisa A; Pearson, Joanna F; Appleby, Peter A; Walker, Dawn; Eardley, Ian; Southgate, Jennifer

2013-08-01

In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca²⁺. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca²⁺ and in a scratch repair assay. The results confirmed the functional expression of P2Y₄ receptors and excluded nonexpressed receptors/channels (P2X₁, P2X₃, P2X₆, P2Y₆, P2Y₁₁, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X₂, P2X₄, P2Y₁, P2Y₂, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca²⁺ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting.

Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium

PubMed Central

Shabir, Saqib; Cross, William; Kirkwood, Lisa A.; Pearson, Joanna F.; Appleby, Peter A.; Walker, Dawn; Eardley, Ian

2013-01-01

In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca2+. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca2+ and in a scratch repair assay. The results confirmed the functional expression of P2Y4 receptors and excluded nonexpressed receptors/channels (P2X1, P2X3, P2X6, P2Y6, P2Y11, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X2, P2X4, P2Y1, P2Y2, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca2+ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting. PMID:23720349

Development of an Efficient Identifier for Nuclear Power Plant Transients Based on Latest Advances of Error Back-Propagation Learning Algorithm

NASA Astrophysics Data System (ADS)

Moshkbar-Bakhshayesh, Khalil; Ghofrani, Mohammad B.

2014-02-01

This study aims to improve the performance of nuclear power plants (NPPs) transients training and identification using the latest advances of error back-propagation (EBP) learning algorithm. To this end, elements of EBP, including input data, initial weights, learning rate, cost function, activation function, and weights updating procedure are investigated and an efficient neural network is developed. Usefulness of modular networks is also examined and appropriate identifiers, one for each transient, are employed. Furthermore, the effect of transient type on transient identifier performance is illustrated. Subsequently, the developed transient identifier is applied to Bushehr nuclear power plant (BNPP). Seven types of the plant events are probed to analyze the ability of the proposed identifier. The results reveal that identification occurs very early with only five plant variables, whilst in the previous studies a larger number of variables (typically 15 to 20) were required. Modular networks facilitated identification due to its sole dependency on the sign of each network output signal. Fast training of input patterns, extendibility for identification of more transients and reduction of false identification are other advantageous of the proposed identifier. Finally, the balance between the correct answer to the trained transients (memorization) and reasonable response to the test transients (generalization) is improved, meeting one of the primary design criteria of identifiers.

Estradiol-induced gene expression in largemouth bass (Micropterus salmoides)

USGS Publications Warehouse

Bowman, C.J.; Kroll, K.J.; Gross, T.G.; Denslow, N.D.

2002-01-01

Vitellogenin (Vtg) and estrogen receptor (ER) gene expression levels were measured in largemouth bass to evaluate the activation of the ER-mediated pathway by estradiol (E2). Single injections of E2 ranging from 0.0005 to 5 mg/kg up-regulated plasma Vtg in a dose-dependent manner. Vtg and ER mRNAs were measured using partial cDNA sequences corresponding to the C-terminal domain for Vtg and the ligand-binding domain of ER?? sequences. After acute E2-exposures (2 mg/kg), Vtg and ER mRNAs and plasma Vtg levels peaked after 2 days. The rate of ER mRNA accumulation peaked 36-42 h earlier than Vtg mRNA. The expression window for ER defines the primary response to E2 in largemouth bass and that for Vtg a delayed primary response. The specific effect of E2 on other estrogen-regulated genes was tested during these same time windows using differential display RT-PCR. Specific up-regulated genes that are expressed in the same time window as Vtg were ERp72 (a membrane-bound disulfide isomerase) and a gene with homology to an expressed gene identified in zebrafish. Genes that were expressed in a pattern that mimics the ER include the gene for zona radiata protein ZP2, and a gene with homology to an expressed gene found in winter flounder. One gene for fibrinogen ?? was down-regulated and an unidentified gene was transiently up-regulated after 12 h of exposure and returned to basal levels by 48 h. Taken together these studies indicate that the acute molecular response to E2 involves a complex network of responses over time. ?? 2002 Elsevier Science Ireland Ltd. All rights reserved.

Functional characterization of two new members of the caffeoyl CoA O-methyltransferase-like gene family from Vanilla planifolia reveals a new class of plastid-localized O-methyltransferases.

PubMed

Widiez, Thomas; Hartman, Thomas G; Dudai, Nativ; Yan, Qing; Lawton, Michael; Havkin-Frenkel, Daphna; Belanger, Faith C

2011-08-01

Caffeoyl CoA O-methyltransferases (OMTs) have been characterized from numerous plant species and have been demonstrated to be involved in lignin biosynthesis. Higher plant species are known to have additional caffeoyl CoA OMT-like genes, which have not been well characterized. Here, we identified two new caffeoyl CoA OMT-like genes by screening a cDNA library from specialized hair cells of pods of the orchid Vanilla planifolia. Characterization of the corresponding two enzymes, designated Vp-OMT4 and Vp-OMT5, revealed that in vitro both enzymes preferred as a substrate the flavone tricetin, yet their sequences and phylogenetic relationships to other enzymes are distinct from each other. Quantitative analysis of gene expression indicated a dramatic tissue-specific expression pattern for Vp-OMT4, which was highly expressed in the hair cells of the developing pod, the likely location of vanillin biosynthesis. Although Vp-OMT4 had a lower activity with the proposed vanillin precursor, 3,4-dihydroxybenzaldehyde, than with tricetin, the tissue specificity of expression suggests it may be a candidate for an enzyme involved in vanillin biosynthesis. In contrast, the Vp-OMT5 gene was mainly expressed in leaf tissue and only marginally expressed in pod hair cells. Phylogenetic analysis suggests Vp-OMT5 evolved from a cyanobacterial enzyme and it clustered within a clade in which the sequences from eukaryotic species had predicted chloroplast transit peptides. Transient expression of a GFP-fusion in tobacco demonstrated that Vp-OMT5 was localized in the plastids. This is the first flavonoid OMT demonstrated to be targeted to the plastids.

Transient gene and microRNA expression profile changes of confluent human fibroblast cells in space

NASA Astrophysics Data System (ADS)

Wu, Honglu; Story, Michael; Karouia, Fathi; Stodieck, Louis; Zhang, Ye; Lu, Tao

2016-07-01

Microgravity, or an altered gravity environment from the Earth1g, has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of these cells. Whether non-proliferating cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted onboard the International Space Station (ISS), confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days, respectively, for investigations of gene and miRNA expression profile changes in these cells. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly, as measured by the percentage of Ki-67 positive cells. Gene and miRNA expression data indicated activation of NFkB and other growth related pathways involving HGF and Vegf along with down regulation of the Let-7 miRNA family. On Day 14 when the cells were mostly non-proliferating, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples with respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeletal changes via immunohistochemistry staining of the cells with antibodies for αa-tubulin and fibronectin showed no difference between flown and ground samples. Taken together, our study suggests that in true non-dividing human fibroblast cells in culture, microgravity experienced in space has little effect on the gene and miRNA expression profiles.

FoxK1 splice variants show developmental stage-specific plasticity of expression with temperature in the tiger pufferfish.

PubMed

Fernandes, Jorge M O; MacKenzie, Matthew G; Kinghorn, James R; Johnston, Ian A

2007-10-01

FoxK1 is a member of the highly conserved forkhead/winged helix (Fox) family of transcription factors and it is known to play a key role in mammalian muscle development and myogenic stem cell function. The tiger pufferfish (Takifugu rubripes) orthologue of mammalian FoxK1 (TFoxK1) has seven exons and is located in a region of conserved synteny between pufferfish and mouse. TFoxK1 is expressed as three alternative transcripts: TFoxK1-alpha, TFoxK1-gamma and TFoxK1-delta. TFoxK1-alpha is the orthologue of mouse FoxK1-alpha, coding for a putative protein of 558 residues that contains the forkhead and forkhead-associated domains typical of Fox proteins and shares 53% global identity with its mammalian homologue. TFoxK1-gamma and TFoxK1-delta arise from intron retention events and these transcripts translate into the same 344-amino acid protein with a truncated forkhead domain. Neither are orthologues of mouse FoxK1-beta. In adult fish, the TFoxK1 splice variants were differentially expressed between fast and slow myotomal muscle, as well as other tissues, and the FoxK1-alpha protein was expressed in myogenic progenitor cells of fast myotomal muscle. During embryonic development, TFoxK1 was transiently expressed in the developing somites, heart, brain and eye. The relative expression of TFoxK1-alpha and the other two alternative transcripts varied with the incubation temperature regime for equivalent embryonic stages and the differences were particularly marked at later developmental stages. The developmental expression pattern of TFoxK1 and its localisation to mononuclear myogenic progenitor cells in adult fast muscle indicate that it may play an essential role in myogenesis in T. rubripes.

Region-specific expression of vesicular glutamate and GABA transporters under various ischaemic conditions in mouse forebrain and retina.

PubMed

Michalski, D; Härtig, W; Krügel, K; Edwards, R H; Böddener, M; Böhme, L; Pannicke, T; Reichenbach, A; Grosche, A

2013-02-12

There is accumulating evidence that glutamate and GABA release are key mechanisms of ischaemic events in the CNS. However, data on the expression of involved transporters for these mediators are inconsistent, potentially impeding further neuroprotective approaches. Here, we applied immunofluorescence labelling to characterise the expression pattern of vesicular glutamate (VGLUT) and GABA transporters (VGAT) after acute focal cerebral ischaemia and in two models of retinal ischaemia. Mice were subjected to filament-based focal cerebral ischaemia predominantly involving the middle cerebral artery territory, also leading to retinal ischaemia due to central retinal artery occlusion (CRAO). Alternatively, retinal ischaemia was induced by a transient increase of the intraocular pressure (HIOP). One day after ischaemia onset, diminished immunolabelling of neuronal nuclei and microtubule-associated protein 2-positive structures were found in the ipsilateral neocortex, subcortex and the retina, indicating neuronal degeneration. VGLUT1 expression did not change significantly in ischaemic tissues whereas VGLUT2 was down-regulated in specific areas of the brain. VGLUT3 expression was only slightly down-regulated in the ischaemia-affected neocortex, and was found to form clusters on fibrils of unknown origin in the ischaemic lateral hypothalamus. In contrast, retinae subjected to CRAO or HIOP displayed a rapid loss of VGLUT3-immunoreactivity. The expression of VGAT appears resistant to ischaemia as there was no significant alteration in all the regions analysed. In summary, these data indicate a region- and subtype-specific change of VGLUT expression in the ischaemia-affected CNS, whose consideration might help to generate specific neuroprotective strategies. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Expression of CTLA-4 (CD152) on human medullary CD4+ thymocytes.

PubMed

Castan, J; Klauenberg, U; Kalmár, P; Fleischer, B; Bröker, B M

1998-06-01

CTLA-4 (CD152) is a T cell surface receptor with sequence homology to the co-stimulatory molecule CD28. The molecule, which is essential for the inhibitory regulation of the immune response, becomes transiently expressed on mature T cells after stimulation in vitro. In situ, CTLA-4+ T cells are enriched in the light zones of the germinal centers in human peripheral lymphoid organs. In this study we have studied expression of CTLA-4 in human thymus in situ. CTLA-4 was expressed on about one third of CD4+/CD8-/CD1- medullary thymocytes. CTLA-4 was acquired by a subset of immature (CD1+) thymocytes and lost from the mature (CD1-) subpopulation within 48 h of cell culture, suggesting that the expression on medullary thymocytes is transient. The demonstration of CTLA-4 on a substantial subpopulation of mature CD4+ thymocytes adds a new dimension to the understanding of this important molecule. When contemplating application of anti-CTLA-4 for therapy its potential influence on T cell maturation has to be taken into account.

Transient Expression of Lumbrokinase (PI239) in Tobacco (Nicotiana tabacum) Using a Geminivirus-Based Single Replicon System Dissolves Fibrin and Blood Clots.

PubMed

Dickey, Alexia; Wang, Nan; Cooper, Edwin; Tull, Lauren; Breedlove, Drew; Mason, Hugh; Liu, Dehu; Wang, Kevin Yueju

2017-01-01

Lumbrokinases, a group of fibrinolytic enzymes extracted from earthworm, have been widely used to prevent and treat various cardiovascular diseases. They specifically target fibrin to effectively degrade thrombi without major side effects. Plant expression systems are becoming potential alternative expression platforms for producing pharmaceutical proteins. In this work, a lumbrokinase (PI239) was produced from a plant system. Both wild-type (WT) and plant codon-optimized (OP) PI239 gene sequences were synthesized and cloned into a geminivirus-based single-vector DNA replicon system. Both vectors were independently expressed in tobacco (Nicotiana tabacum) leaves transiently by agroinfiltration. Overexpressed PI239 resulted in sudden tissue necrosis 3 days after infiltration. Remaining proteins were purified through His-tag affinity chromatography and analyzed with SDS-PAGE and Western blot methods. Purified PI239 successfully degraded artificial fibrin with relative activity of 13,400 U/mg when compared with commercial lumbrokinase product. In vitro tests demonstrated that plant-derived PI239 dissolved human blood clots and that the plant expression system is capable of producing functional PI239.

Phytochromes A and C cooperatively regulate early and transient gene expression after red-light irradiation in rice seedlings.

PubMed

Kiyota, Seiichiro; Xie, Xianzhi; Takano, Makoto

2012-02-01

Phytochromes are red/far-red photoreceptors encoded by a small gene family in higher plants. Differences in phenotype among mutants suggest distinct functions among phytochrome subfamilies. We attempted to find distinct functions among phytochromes by oligo-microarray analysis of single, double, and triple mutants in rice. In most cases, gene expression was redundantly regulated by phytochromes A and B after irradiation by a red light pulse in etiolated rice shoots. However, we found that several genes were specifically regulated by phytochromes A and C. Most of them were expressed immediately after the red light pulse in a transient manner. They are stress-related genes that may be involved in resistance to light stress when etiolated seedlings are exposed to light. These genes were not expressed in green leaves after the red light pulse, suggesting that they have a function specific to etiolated seedlings. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Leaf-Encapsulated Vaccines: Agroinfiltration and Transient Expression of the Antigen Staphylococcal Endotoxin B in Radish Leaves

PubMed Central

Liu, Pei-Feng; Wang, Yanhan; Ulrich, Robert G.; Simmons, Christopher W.; VanderGheynst, Jean S.; Gallo, Richard L.

2018-01-01

Transgene introgression is a major concern associated with transgenic plant-based vaccines. Agroinfiltration can be used to selectively transform nonreproductive organs and avoid introgression. Here, we introduce a new vaccine modality in which Staphylococcal enterotoxin B (SEB) genes are agroinfiltrated into radishes (Raphanw sativus L.), resulting in transient expression and accumulation of SEB in planta. This approach can simultaneously express multiple antigens in a single leaf. Furthermore, the potential of high-throughput vaccine production was demonstrated by simultaneously agroinfiltrating multiple radish leaves using a multichannel pipette. The expression of SEB was detectable in two leaf cell types (epidermal and guard cells) in agroinfiltrated leaves. ICR mice intranasally immunized with homogenized leaves agroinfiltrated with SEB elicited detectable antibody to SEB and displayed protection against SEB-induced interferon-gamma (IFN-γ) production. The concept of encapsulating antigens in leaves rather than purifying them for immunization may facilitate rapid vaccine production during an epidemic disease. PMID:29577048

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

PubMed

Quaedvlieg, N E; Schlaman, H R; Admiraal, P C; Wijting, S E; Stougaard, J; Spaink, H P

1998-07-01

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

PubMed

Quaedvlieg, N E; Schlaman, H R; Admiraal, P C; Wijting, S E; Stougaard, J; Spaink, H P

1998-11-01

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Human Neural Cells Transiently Express Reelin during Olfactory Placode Development

PubMed Central

Antal, M. Cristina; Samama, Brigitte; Ghandour, M. Said; Boehm, Nelly

2015-01-01

Reelin, an extracellular glycoprotein is essential for migration and correct positioning of neurons during development. Since the olfactory system is known as a source of various migrating neuronal cells, we studied Reelin expression in the two chemosensory olfactory systems, main and accessory, during early developmental stages of human foetuses/embryos from Carnegie Stage (CS) 15 to gestational week (GW) 14. From CS 15 to CS 18, but not at later stages, a transient expression of Reelin was detected first in the presumptive olfactory and then in the presumptive vomeronasal epithelium. During the same period, Reelin-positive cells detach from the olfactory/vomeronasal epithelium and migrate through the mesenchyme beneath the telencephalon. Dab 1, an adaptor protein of the Reelin pathway, was simultaneously expressed in the migratory mass from CS16 to CS17 and, at later stages, in the presumptive olfactory ensheathing cells. Possible involvements of Reelin and Dab 1 in the peripheral migrating stream are discussed. PMID:26270645

A simple method to generate stable cell lines for the analysis of transient protein-protein interactions.

PubMed

Savage, Emilia Elizabeth; Wootten, Denise; Christopoulos, Arthur; Sexton, Patrick Michael; Furness, Sebastian George Barton

2013-04-01

Transient protein-protein interactions form the basis of signal transduction pathways in addition to many other biological processes. One tool for studying these interactions is bioluminescence resonance energy transfer (BRET). This technique has been widely applied to study signaling pathways, in particular those initiated by G protein-coupled receptors (GPCRs). These assays are routinely performed using transient transfection, a technique that has limitations in terms of assay cost and variability, overexpression of interacting proteins, vector uptake limited to cycling cells, and non-homogenous expression across cells within the assay. To address these issues, we developed bicistronic vectors for use with Life Technology's Gateway and flpIN systems. These vectors provide a means to generate isogenic cell lines for comparison of interacting proteins. They have the advantage of stable, single copy, isogenic, homogeneous expression with low inter-assay variation. We demonstrate their utility by assessing ligand-induced interactions between GPCRs and arrestin proteins.

Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling.

PubMed

Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

2015-09-15

Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system.

Review and Extension of CO2-Based Methods to Determine Ventilation Rates with Application to School Classrooms

PubMed Central

Batterman, Stuart

2017-01-01

The ventilation rate (VR) is a key parameter affecting indoor environmental quality (IEQ) and the energy consumption of buildings. This paper reviews the use of CO2 as a “natural” tracer gas for estimating VRs, focusing on applications in school classrooms. It provides details and guidance for the steady-state, build-up, decay and transient mass balance methods. An extension to the build-up method and an analysis of the post-exercise recovery period that can increase CO2 generation rates are presented. Measurements in four mechanically-ventilated school buildings demonstrate the methods and highlight issues affecting their applicability. VRs during the school day fell below recommended minimum levels, and VRs during evening and early morning were on the order of 0.1 h−1, reflecting shutdown of the ventilation systems. The transient mass balance method was the most flexible and advantageous method given the low air change rates and dynamic occupancy patterns observed in the classrooms. While the extension to the build-up method improved stability and consistency, the accuracy of this and the steady-state method may be limited. Decay-based methods did not reflect the VR during the school day due to heating, ventilation and air conditioning (HVAC) system shutdown. Since the number of occupants in classrooms changes over the day, the VR expressed on a per person basis (e.g., L·s−1·person−1) depends on the occupancy metric. If occupancy measurements can be obtained, then the transient mass balance method likely will provide the most consistent and accurate results among the CO2-based methods. Improved VR measurements can benefit many applications, including research examining the linkage between ventilation and health. PMID:28165398

Review and Extension of CO₂-Based Methods to Determine Ventilation Rates with Application to School Classrooms.

PubMed

Batterman, Stuart

2017-02-04

The ventilation rate (VR) is a key parameter affecting indoor environmental quality (IEQ) and the energy consumption of buildings. This paper reviews the use of CO₂ as a "natural" tracer gas for estimating VRs, focusing on applications in school classrooms. It provides details and guidance for the steady-state, build-up, decay and transient mass balance methods. An extension to the build-up method and an analysis of the post-exercise recovery period that can increase CO₂ generation rates are presented. Measurements in four mechanically-ventilated school buildings demonstrate the methods and highlight issues affecting their applicability. VRs during the school day fell below recommended minimum levels, and VRs during evening and early morning were on the order of 0.1 h -1 , reflecting shutdown of the ventilation systems. The transient mass balance method was the most flexible and advantageous method given the low air change rates and dynamic occupancy patterns observed in the classrooms. While the extension to the build-up method improved stability and consistency, the accuracy of this and the steady-state method may be limited. Decay-based methods did not reflect the VR during the school day due to heating, ventilation and air conditioning (HVAC) system shutdown. Since the number of occupants in classrooms changes over the day, the VR expressed on a per person basis (e.g., L·s -1 ·person -1 ) depends on the occupancy metric. If occupancy measurements can be obtained, then the transient mass balance method likely will provide the most consistent and accurate results among the CO₂-based methods. Improved VR measurements can benefit many applications, including research examining the linkage between ventilation and health.

Numerical Investigation of Pre-detonator Geometries for PDE Applications

DTIC Science & Technology

2010-03-01

pattern will be traced that resembles the scales of a fish. The fishscale pattern is a trace of the high pressure transverse waves. Figure 10 shows a...numerically generated smokefoil record that is created by taking a peak pressure histogram of the transient data set. Each of the fishscale -like diamonds

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

PubMed

Fabrick, Jeffrey A; Hull, J Joe

2017-04-20

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment.

PubMed Central

Lopata, M A; Cleveland, D W; Sollner-Webb, B

1984-01-01

Using a plasmid containing the bacterial chloramphenicol acetyl transferase gene, we have assayed for transient expression of DNA introduced into mouse L cells by a variety of transfection conditions. High efficiency uptake and expression of this foreign DNA have been achieved by modifying the DEAE dextran mediated transfection procedure of McCutchan and Pagano (1) to include a shock with either dimethyl sulfoxide or glycerol. Inclusion of the shock step can increase expression of the transfected gene a surprising approximately 50 fold. With plasmid constructs that do not replicate after transfection, we can readily detect CAT activity in an overnight autoradiographic exposure from less than 0.1% of an extract from a 60 mm dish of transfected cells. We have determined the amounts of DNA, the amount and time course of DEAE-dextran and dimethyl sulfoxide treatments, the effects of additional DNA, and the time after transfection which yield maximal expression. Overall, this transfection protocol using DEAE-dextran coupled to a shock treatment is simple, straightforward, and gives consistently high levels of expression of the input DNA. Images PMID:6589587

Isothiocyanates from Wasabia japonica activate transient receptor potential ankyrin 1 channel.

PubMed

Uchida, Kunitoshi; Miura, Yosuke; Nagai, Masashi; Tominaga, Makoto

2012-11-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) and 6-(methylthio)hexyl isothiocyanate (6-MTITC) have low pungency and are responsible for the fresh flavor of wasabi (Wasabia japonica [Miq] Matsumura). In this study, we found that these two isothiocyanates activate transient receptor potential ankyrin 1 (TRPA1), and 6-MSITC activates transient receptor potential vanilloid 1 (TRPV1), but not other transient receptor potential channels expressed in sensory neurons. Both 6-MSITC and 6-MTITCinduced intracellular Ca(2+) increases in human embryonic kidney-derived 293 cells expressing mouse TRPA1 (mTRPA1) as measured by Ca(2+) imaging. In whole-cell patch-clamp recordings, 6-MSITC and 6-MTITC dose-dependently activated both mTRPA1 (EC(50) = 147±26 µM for 6-MSITC and 30±3 µM for 6-MTITC) and human TRPA1 (hTRPA1; EC(50) = 39±4 µM for 6-MSITC and 34±3 µM for 6-MTITC). In addition, TRPA1 N-terminal cysteines, which are reported to be important for channel activation by electrophilic ligands, were involved in 6-MSITC- and 6-MTITC-evoked TRPA1 activation. These isothiocyanates also activated endogenous TRPA1 expressed in mouse dorsal root ganglion neurons and intraplantar injection of 10-30 mM 6-MSITC-evoked pain-related behaviors in mice. These results indicate the following: 1) 6-MSITC and 6-MTITC activate both mTRPA1 and hTRPA1; 2) 6-MSITC activates mTRPV1; and 3) the pharmacological functions of these isothiocyanates could be derived from TRPA1 activation.

Generalization of Wilemski-Fixman-Weiss decoupling approximation to the case involving multiple sinks of different sizes, shapes, and reactivities.

PubMed

Uhm, Jesik; Lee, Jinuk; Eun, Changsun; Lee, Sangyoub

2006-08-07

We generalize the Wilemski-Fixman-Weiss decoupling approximation to calculate the transient rate of absorption of point particles into multiple sinks of different sizes, shapes, and reactivities. As an application we consider the case involving two spherical sinks. We obtain a Laplace-transform expression for the transient rate that is in excellent agreement with computer simulations. The long-time steady-state rate has a relatively simple expression, which clearly shows the dependence on the diffusion constant of the particles and on the sizes and reactivities of sinks, and its numerical result is in good agreement with the known exact result that is given in terms of recursion relations.

Concurrent Transient Activation of Wnt/{beta}-Catenin Pathway Prevents Radiation Damage to Salivary Glands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hai Bo; Yang Zhenhua; Shangguan Lei

2012-05-01

Purpose: Many head and neck cancer survivors treated with radiotherapy suffer from permanent impairment of their salivary gland function, for which few effective prevention or treatment options are available. This study explored the potential of transient activation of Wnt/{beta}-catenin signaling in preventing radiation damage to salivary glands in a preclinical model. Methods and Materials: Wnt reporter transgenic mice were exposed to 15 Gy single-dose radiation in the head and neck area to evaluate the effects of radiation on Wnt activity in salivary glands. Transient Wnt1 overexpression in basal epithelia was induced in inducible Wnt1 transgenic mice before together with, after,more » or without local radiation, and then saliva flow rate, histology, apoptosis, proliferation, stem cell activity, and mRNA expression were evaluated. Results: Radiation damage did not significantly affect activity of Wnt/{beta}-catenin pathway as physical damage did. Transient expression of Wnt1 in basal epithelia significantly activated the Wnt/{beta}-catenin pathway in submandibular glands of male mice but not in those of females. Concurrent transient activation of the Wnt pathway prevented chronic salivary gland dysfunction following radiation by suppressing apoptosis and preserving functional salivary stem/progenitor cells. In contrast, Wnt activation 3 days before or after irradiation did not show significant beneficial effects, mainly due to failure to inhibit acute apoptosis after radiation. Excessive Wnt activation before radiation failed to inhibit apoptosis, likely due to extensive induction of mitosis and up-regulation of proapoptosis gene PUMA while that after radiation might miss the critical treatment window. Conclusion: These results suggest that concurrent transient activation of the Wnt/{beta}-catenin pathway could prevent radiation-induced salivary gland dysfunction.« less

Postischemic alterations of BDNF, NGF, HSP 70 and ubiquitin immunoreactivity in the gerbil hippocampus: pharmacological approach.

PubMed

Himeda, Toshiki; Tounai, Hiroko; Hayakawa, Natsumi; Araki, Tsutomu

2007-03-01

1. We investigated the immunohistochemical alterations of BDNF, NGF, HSP 70 and ubiquitin in the hippocampus 1 h to 14 days after transient cerebral ischemia in gerbils. We also examined the effect of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor pitavastatin against the changes of BDNF, NGF, HSP 70 and ubiquitin in the hippocampus after cerebral ischemia in the hippocampus after ischemia. 2. The transient cerebral ischemia was carried out by clamping the carotid arteries with aneurismal clips for 5 min. 3. In the present study, the alteration of HSP 70 and ubiquitin immunoreactivity in the hippocampal CA1 sector was more pronounced than that of BDNF and NGF immunoreactivity after transient cerebral ischemia. In double-labeled immunostainings, BDNF, NGF and ubiquitin immunostaining was observed both in GFAP-positive astrocytes and MRF-1-positive microglia in the hippocampal CA1 sector after ischemia. Furthermore, prophylactic treatment with pitavastatin prevented the damage of neurons with neurotrophic factor and stress proteins in the hippocampal CA1 sector after ischemia. 4. These findings suggest that the expression of stress protein including HSP 70 and ubiquitin may play a key role in the protection against the hippocampal CA1 neuronal damage after transient cerebral ischemia in comparison with the expression of neurotrophic factor such as BDNF and NGF. The present findings also suggest that the glial BDNF, NGF and ubiquitin may play some role for helping surviving neurons after ischemia. Furthermore, our present study indicates that prophylactic treatment with pitavastatin can prevent the damage of neurons with neurotrophic factor and stress proteins in the hippocampal CA1 sector after transient cerebral ischemia. Thus our study provides further valuable information for the pathogenesis after transient cerebral ischemia.

Severe hydramnios and preterm delivery in association with transient maternal diabetes insipidus.

PubMed

Weinberg, Lori E; Dinsmoor, Mara J; Silver, Richard K

2010-08-01

Diabetes insipidus is rare in pregnancy. It is characterized by hypoosmolar polyuria and may be central, nephrogenic, or transient in etiology; the latter is presumably related to excess placental vasopresinase production. In theory, fetal effects of this endocrine condition may include hydramnios secondary to fetal polyuria. A pregnant patient developed rapid-onset second-trimester hydramnios that prompted a thorough fetal and maternal evaluation. She ultimately was diagnosed with transient diabetes insipidus of pregnancy because of an abrupt change in her voiding pattern at 20 weeks of gestation, significant polydipsia, and laboratory studies that revealed a hypoosmolar polyuria with normal serum and urine electrolytes. Transient neonatal polyuria also was confirmed in association with this unique maternal endocrine syndrome. The most likely cause of hydramnios in this case is transient maternal diabetes insipidus of pregnancy from excessive secretion of placental vasopressinase resulting in fetal polyuria. In cases of hydramnios of unknown etiology, if a history of maternal polyuria is elicited and confirmed, diabetes insipidus of pregnancy may play a role in some cases.

Transient bedrock channel evolution across a precipitation gradient: A case study from Kohala, Hawaii.

NASA Astrophysics Data System (ADS)

Gasparini, N. M.; Han, J.; Johnson, J. P.; Menking, J. A.

2011-12-01

This study uses observations from the Kohala Peninsula, on the Big Island of Hawaii, and numerical modeling, to explore how precipitation gradients may affect fluvial bedrock incision and channel morphology. Orographic precipitation patterns result in over 4 m/yr of rainfall on the wet side of the peninsula and less than 0.5 m/yr on the dry side. These precipitation patterns likely strongly contribute to the observed channel morphology. Further, the region is subsiding, leading to prolonged transient channel evolution. We explore changes in a number of channel morphologic parameters with watershed averaged precipitation rate. We use PRISM precipitation data and data from isohyets developed from historic rain gauge data. Not surprisingly, valley depth, measured from a 10 meter DEM, increases with spatially averaged precipitation rate. We also find that channel profile form varies with precipitation rate, with drier channels exhibiting a straight to slightly concave channel form and wetter channels exhibiting a convex to concave channel form. The precipitation value at which this transition in channel profile form occurs depends on the precipitation data-set used, highlighting the need for more accurate measurements of precipitation in settings with extreme precipitation patterns similar to our study area. The downstream pattern in precipitation is likely significant in the development of the convex-concave profile form. Numerical modeling results support that precipitation patterns such as those observed on the wet-side of the Kohala Peninsula may contribute to the convex-concave profile form. However, we emphasize that while precipitation patterns may contribute to the channel form, these channel features are transient and not expected to be sustained in steady-state landscapes. We also emphasize that it is fluvial discharge, as driven by precipitation, rather than precipitation alone, that drives the processes shaping the channel form. Because fluvial discharge is integrative, relatively extreme precipitation gradients are required to produce anomalous channel profile forms.

Dynamic characteristics of a pump-turbine during hydraulic transients of a model pumped-storage system: 3D CFD simulation

NASA Astrophysics Data System (ADS)

Zhang, X. X.; Cheng, Y. G.; Xia, L. S.; Yang, J. D.

2014-03-01

The runaway process in a model pumped-storage system was simulated for analyzing the dynamic characteristics of a pump-turbine. The simulation was adopted by coupling 1D (One Dimensional) pipeline MOC (Method of Characteristics) equations with a 3D (Three Dimensional) pump-turbine CFD (Computational Fluid Dynamics) model, in which the water hammer wave in the 3D zone was defined by giving a pressure dependent density. We found from the results that the dynamic performances of the pump-turbine do not coincide with the static operating points, especially in the S-shaped characteristics region, where the dynamic trajectories follow ring-shaped curves. Specifically, the transient operating points with the same Q11 and M11 in different moving directions of the dynamic trajectories give different n11. The main reason of this phenomenon is that the transient flow patterns inside the pump-turbine are influenced by the ones in the previous time step, which leads to different flow patterns between the points with the same Q11 and M11 in different moving directions of the dynamic trajectories.

Interictal spike frequency varies with ovarian cycle stage in a rat model of epilepsy.

PubMed Central

D’Amour, James; Magagna-Poveda, Alejandra; Moretto, Jillian; Friedman, Daniel; LaFrancois, John J.; Pearce, Patrice; Fenton, Andre A.; MacLusky, Neil J.; Scharfman, Helen E.

2015-01-01

In catamenial epilepsy, seizures exhibit a cyclic pattern that parallels the menstrual cycle. Many studies suggest that catamenial seizures are caused by fluctuations in gonadal hormones during the menstrual cycle, but this has been difficult to study in rodent models of epilepsy because the ovarian cycle in rodents, called the estrous cycle, is disrupted by severe seizures. Thus, when epilepsy is severe, estrous cycles become irregular or stop. Therefore, we modified kainic acid (KA)- and pilocarpine-induced status epilepticus (SE) models of epilepsy so that seizures were rare for the first months after SE, and conducted video-EEG during this time. The results showed that interictal spikes (IIS) occurred intermittently. All rats with regular 4-day estrous cycles had IIS that waxed and waned with the estrous cycle. The association between the estrous cycle and IIS was strong: if the estrous cycles became irregular transiently, IIS frequency also became irregular, and when the estrous cycle resumed its 4-day pattern, IIS frequency did also. Furthermore, when rats were ovariectomized, or males were recorded, IIS frequency did not show a 4-day pattern. Systemic administration of an estrogen receptor antagonist stopped the estrous cycle transiently, accompanied by transient irregularity of the IIS pattern. Eventually all animals developed severe, frequent seizures and at that time both the estrous cycle and the IIS became irregular. We conclude that the estrous cycle entrains IIS in the modified KA and pilocarpine SE models of epilepsy. The data suggest that the ovarian cycle influences more aspects of epilepsy than seizure susceptibility. PMID:25864929

Architectural patterns of p16 immunohistochemical expression associated with cancer immunity and prognosis of head and neck squamous cell carcinoma.

PubMed

Ryu, Hyang Joo; Kim, Eun Kyung; Heo, Su Jin; Cho, Byoung Chul; Kim, Hye Ryun; Yoon, Sun Och

2017-11-01

We evaluated the expression patterns of p16, which is used as a surrogate marker of HPV infection in head and neck squamous cell carcinoma (HNSCC), in regard to their biological and prognostic implications. p16 expression patterns and infiltrated immune cells were analyzed through immunohistochemistry of p16, CD3, CD8, PD-1, FOXP3, and CD163 on surgically resected HNSCCs (n = 393). Patterns of p16 immunoexpression were defined as STRONG (strong, diffuse expression in cytoplasm, and nucleus in >70% of tumor cells), MARGINAL (expression restricted to tumor margins), MOSAIC (ragged, discontinued expression), NUCLEAR (expression in nuclei only), and ABSENT (no expression). The STRONG pattern was more frequent in the oropharynx, and the MARGINAL pattern was noted only in the oral cavity. MOSAIC and NUCLEAR patterns were noted at variable sites. No two patterns of p16 expression showed the same immune cell composition of CD3+ T cells, CD8+ cytotoxic T cells, PD-1+ T cells, FOXP3+ regulatory T cells, and CD163+ macrophages. In overall and disease-free survival analyses, the STRONG pattern showed the most favorable prognosis, while the NUCLEAR pattern had the worst prognosis. HNSCC anatomical sites, tumor-related immune cell components, and patient outcomes were associated with p16 expression patterns. Each architectural pattern of p16 expression may be related to different biological and prognostic phenotypes. © 2017 APMIS. Published by John Wiley & Sons Ltd.

An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles.

PubMed

Santi, Luca; Batchelor, Lance; Huang, Zhong; Hjelm, Brooke; Kilbourne, Jacquelyn; Arntzen, Charles J; Chen, Qiang; Mason, Hugh S

2008-03-28

Virus-like particles (VLPs) derived from enteric pathogens like Norwalk virus (NV) are well suited to study oral immunization. We previously described stable transgenic plants that accumulate recombinant NV-like particles (rNVs) that were orally immunogenic in mice and humans. The transgenic approach suffers from long generation time and modest level of antigen accumulation. We now overcome these constraints with an efficient tobacco mosaic virus (TMV)-derived transient expression system using leaves of Nicotiana benthamiana. We produced properly assembled rNV at 0.8 mg/g leaf 12 days post-infection (dpi). Oral immunization of CD1 mice with 100 or 250 microg/dose of partially purified rNV elicited systemic and mucosal immune responses. We conclude that the plant viral transient expression system provides a robust research tool to generate abundant quantities of rNV as enriched, concentrated VLP preparations that are orally immunogenic.

An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles

PubMed Central

Santi, Luca; Batchelor, Lance; Huang, Zhong; Hjelm, Brooke; Kilbourne, Jacquelyn; Arntzen, Charles J.; Chen, Qiang; Mason, Hugh S.

2009-01-01

Virus like particles (VLPs) derived from enteric pathogens like Norwalk virus (NV) are well suited to study oral immunization. We previously described stable transgenic plants that accumulate recombinant NV-like particles (rNV) that were orally immunogenic in mice and humans. The transgenic approach suffers from long generation time and modest level of antigen accumulation. We now overcome these constraints with an efficient tobacco mosaic virus (TMV)-derived transient expression system using leaves of Nicotiana benthamiana. We produced properly assembled rNV at 0.8 mg/g leaf 12 days post infection. Oral immunization of CD1 mice with 100 or 250 μg/dose of partially purified rNV elicited systemic and mucosal immune responses. We conclude that the plant viral transient expression system provides a robust research tool to generate abundant quantities of rNV as enriched, concentrated VLP preparations that are orally immunogenic. PMID:18325641

Transient receptor potential channel M5 and phospholipaseC-beta2 colocalizing in zebrafish taste receptor cells.

PubMed

Yoshida, Yuki; Saitoh, Kana; Aihara, Yoshiko; Okada, Shinji; Misaka, Takumi; Abe, Keiko

2007-10-08

In mammals, transient receptor potential (TRP) channel M5 (TRPM5) is coexpressed with phospholipaseC-beta2 (PLC-beta2) in the taste receptor cells, and both PLC-beta2 and TRPM5 are essential elements in the signal transduction of sweet, bitter and umami stimuli. In this study, we identified the zebrafish homologue of TRPM5 (zfTRPM5) and examined its expression in the gustatory system by in-situ hybridization. Using a transgenic zebrafish line that expressed green fluorescent protein under the control of the PLC-beta2 promoter, we showed that zfTRPM5 is expressed in green fluorescent protein-labeled cells of the taste buds. These results demonstrate that zfTRPM5 and PLC-beta2 colocalize in zebrafish taste receptor cells, suggesting their crucial roles in taste signaling via the fish taste receptors.

Canonical transient receptor potential channel 2 (TRPC2): old name-new games. Importance in regulating of rat thyroid cell physiology.

PubMed

Törnquist, Kid; Sukumaran, Pramod; Kemppainen, Kati; Löf, Christoffer; Viitanen, Tero

2014-11-01

In addition to the TSH-cyclic AMP signalling pathway, calcium signalling is of crucial importance in thyroid cells. Although the importance of calcium signalling has been thoroughly investigated for several decades, the nature of the calcium channels involved in signalling is unknown. In a recent series of investigations using the well-studied rat thyroid FRTL-5 cell line, we showed that these cells exclusively express the transient receptor potential canonical 2 (TRPC2) channel. Our results suggested that the TRPC2 channel is of significant importance in regulating thyroid cell function. These investigations were the first to show that thyroid cells express a member of the TRPC family of ion channels. In this review, we will describe the importance of the TRPC2 channel in regulating TSH receptor expression, thyroglobulin maturation, intracellular calcium and iodide homeostasis and that the channel also regulates thyroid cell proliferation.

Cytoskeletal self-organization in neuromorphogenesis.

PubMed

Dehmelt, Leif

2014-01-01

Self-organization of dynamic microtubules via interactions with associated motors plays a critical role in spindle formation. The microtubule-based mechanisms underlying other aspects of cellular morphogenesis, such as the formation and development of protrusions from neuronal cells is less well understood. In a recent study, we investigated the molecular mechanism that underlies the massive reorganization of microtubules induced in non-neuronal cells by expression of the neuronal microtubule stabilizer MAP2c. In that study we directly observed cortical dynein complexes and how they affect the dynamic behavior of motile microtubules in living cells. We found that stationary dynein complexes transiently associate with motile microtubules near the cell cortex and that their rapid turnover facilitates efficient microtubule transport. Here, we discuss our findings in the larger context of cellular morphogenesis with specific focus on self-organizing principles from which cellular shape patterns such as the thin protrusions of neurons can emerge.

Mutism and auditory agnosia due to bilateral insular damage--role of the insula in human communication.

PubMed

Habib, M; Daquin, G; Milandre, L; Royere, M L; Rey, M; Lanteri, A; Salamon, G; Khalil, R

1995-03-01

We report a case of transient mutism and persistent auditory agnosia due to two successive ischemic infarcts mainly involving the insular cortex on both hemispheres. During the 'mutic' period, which lasted about 1 month, the patient did not respond to any auditory stimuli and made no effort to communicate. On follow-up examinations, language competences had re-appeared almost intact, but a massive auditory agnosia for non-verbal sounds was observed. From close inspection of lesion site, as determined with brain resonance imaging, and from a study of auditory evoked potentials, it is concluded that bilateral insular damage was crucial to both expressive and receptive components of the syndrome. The role of the insula in verbal and non-verbal communication is discussed in the light of anatomical descriptions of the pattern of connectivity of the insular cortex.

Cytoskeletal self-organization in neuromorphogenesis

PubMed Central

Dehmelt, Leif

2014-01-01

Self-organization of dynamic microtubules via interactions with associated motors plays a critical role in spindle formation. The microtubule-based mechanisms underlying other aspects of cellular morphogenesis, such as the formation and development of protrusions from neuronal cells is less well understood. In a recent study, we investigated the molecular mechanism that underlies the massive reorganization of microtubules induced in non-neuronal cells by expression of the neuronal microtubule stabilizer MAP2c. In that study we directly observed cortical dynein complexes and how they affect the dynamic behavior of motile microtubules in living cells. We found that stationary dynein complexes transiently associate with motile microtubules near the cell cortex and that their rapid turnover facilitates efficient microtubule transport. Here, we discuss our findings in the larger context of cellular morphogenesis with specific focus on self-organizing principles from which cellular shape patterns such as the thin protrusions of neurons can emerge. PMID:24847718

A novel ethylene responsive factor CitERF13 plays a role in photosynthesis regulation.

PubMed

Xie, Xiu-Lan; Xia, Xiao-Jian; Kuang, Sheng; Zhang, Xi-Li; Yin, Xue-Ren; Yu, Jing-Quan; Chen, Kun-Song

2017-03-01

Ethylene responsive factors (ERFs) act as critical downstream components of the ethylene signalling pathway in regulating plant development and stress responses. However little is known about its role in regulation of photosynthesis. Here, we identified an ethylene-inducible ERF gene in citrus, CitERF13. Transient over-expression of CitERF13 in N. tabacum leaves, resulted in a significant decrease in net photosynthetic rate. Closer examination of photosynthetic activity of PSII and PSI indicated that CitERF13 overexpression led to declines of F v /F m , Y(II) and Y(I). However, change in NPQ was less pronounced. CitERF13 overexpression also significantly reduced V c,max , J max and AQY, indicating inhibition of the Calvin cycle. The expression of photosynthesis-related genes was suppressed to a variable extent in leaf blades transiently over-expressing CitERF13. CitERF13 transient overexpression in tobacco or citrus both resulted in a decline of Chlorophyll content and CitERF13 overexpressing tobacco leaf disc was more susceptible to chlorosis in response to MV-mediated oxidative stress. The results suggest that CitERF13 is potentially involved in suppressing photosynthesis through multiple pathways, for instance, inhibiting photochemical activity of photosynthesis, CO 2 carboxylation capacity and chlorophyll metabolism. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Genetic Transformation of Hordeum vulgare ssp. spontaneum for the Development of a Transposon-Based Insertional Mutagenesis System.

PubMed

Cardinal, Marie-Josée; Kaur, Rajvinder; Singh, Jaswinder

2016-10-01

Domestication and intensive selective breeding of plants has triggered erosion of genetic diversity of important stress-related alleles. Researchers highlight the potential of using wild accessions as a gene source for improvement of cereals such as barley, which has major economic and social importance worldwide. Previously, we have successfully introduced the maize Ac/Ds transposon system for gene identification in cultivated barley. The objective of current research was to investigate the response of Hordeum vulgare ssp. spontaneum wild barley accessions in tissue culture to standardize parameters for introduction of Ac/Ds transposons through genetic transformation. We investigated the response of ten wild barley genotypes for callus induction, regenerative green callus induction and regeneration of fertile plants. The activity of exogenous Ac/Ds elements was observed through a transient assay on immature wild barley embryos/callus whereby transformed embryos/calli were identified by the expression of GUS. Transient Ds expression bombardment experiments were performed on 352 pieces of callus (3-5 mm each) or immature embryos in 4 genotypes of wild barley. The transformation frequency of putative transgenic callus lines based on transient GUS expression ranged between 72 and100 % in wild barley genotypes. This is the first report of a transformation system in H. vulgare ssp. spontaneum.

Transient Overexpression of Gremlin Results in Epithelial Activation and Reversible Fibrosis in Rat Lungs

PubMed Central

Farkas, Laszlo; Farkas, Daniela; Gauldie, Jack; Warburton, David; Shi, Wei; Kolb, Martin

2011-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease of the lung parenchyma, without curative treatment. Gremlin is a bone morphogenic protein (BMP) antagonist, its expression being increased in IPF lungs. It has been implicated in promoting myofibroblast accumulation, likely through inhibited fibroblast apoptosis and epithelial-to-mesenchymal transition. In the current study, we examined the effects of selective adenovirus-mediated overexpression of Gremlin in rat lungs. We show that transient Gremlin overexpression results in activation of alveolar epithelial cells with proliferation and apoptosis, as well as partly reversible lung fibrosis. We found myofibroblasts arranged in fibroblastic foci. Fibroblast proliferation occurred delayed as compared with epithelial changes. Fibrotic pathology significantly declined after Day 14, the reversal being associated with an increase of the epithelium-protective element, fibroblast growth factor (FGF)–10. Our data indicate that Gremlin-mediated BMP inhibition results in activation of epithelial cells and transient fibrosis, but also induction of epithelium-protective FGF10. A Gremlin–BMP–FGF10 loop may explain these results, and demonstrate that the interactions between different factors are quite complex in fibrotic lung disease. Increased Gremlin expression in human IPF tissue may be an expression of continuing epithelial injury, and Gremlin may be part of activated repair mechanisms. PMID:20705941

Relationship of calcitonin mRNA expression to the differentiation state of HL 60 cells.

PubMed

Kiefer, P; Bacher, M; Pflüger, K H

1994-05-01

Raised plasma levels of immunoreactive human calcitonin (ihCT) can be found in patients with myeloid leukemia and seem to indicate a poor prognosis. High levels were found in acute undifferentiated and acute myeloblastic leukemia. To test whether CT expression could be a marker of myeloid differentiation, we used the promyelocytic leukemia cell line HL 60 which also expresses ihCT as a model system for myeloid differentiation. Exponentially growing HL 60 cells as well as differentiation induced HL 60 cells expressed a single 1.0 Kb CT transcript. The induction of HL 60 cell differentiation along the granulocytic lineage by DMSO or HMBA had no effect on the level of CT transcripts. Induction of monocytic/macrophagic differentiation by TPA resulted in a transient, about 10-fold elevated expression of CT steady state mRNA after 24 h. In contrast to TPA, induction of HL 60 cell differentiation along the monocytic pathway by Vit D3 had no detectable effect on the level of the CT in RNA expression at corresponding time points. These findings suggest that the transient induction of CT steady state mRNA expression by TPA is rather a direct effect of the phorbol ester than commitment along the monocytic line of differentiation.

Effects of transient high temperature treatment on the intestinal flora of the silkworm Bombyx mori.

PubMed

Sun, Zhenli; Kumar, Dhiraj; Cao, Guangli; Zhu, Liyuan; Liu, Bo; Zhu, Min; Liang, Zi; Kuang, Sulan; Chen, Fei; Feng, Yongjie; Hu, Xiaolong; Xue, Renyu; Gong, Chengliang

2017-06-13

The silkworm Bombyx mori is a poikilotherm and is therefore sensitive to various climatic conditions. The influence of temperature on the intestinal flora and the relationship between the intestinal flora and gene expression in the silkworm remain unknown. In the present study, changes of the intestinal flora at 48, 96 and 144 h following transient high temperature treatment (THTT) of 37 °C for 8 h were investigated. According to principal component analysis, the abundances of Enterococcus and Staphylococcus showed a negative correlation with other dominant genera. After THTT, the gene expression levels of spatzle-1 and dicer-2 were increased and decreased, respectively, which suggested that the Toll and RNAi pathways were activated and suppressed, respectively. The species-gene expression matrix confirmed that the spatzle-1 and dicer-2 gene expression levels were negatively and positively correlated, respectively, with the abundance of Enterococcus and Staphylococcus in the control. The abundance of Variovorax post-THTT was positively correlated with the spatzle-1 gene expression level, whereas the community richness of Enterococcus was negatively correlated with the spatzle-1 gene expression level and positively correlated with the dicer-2. The results of the present investigation provide new evidence for understanding the relationships among THTT, intestinal flora and host gene expression.

Expression and diagnosis of transient receptor potential vanilloid1 in urothelium of patients with overactive bladder.

PubMed

Zhang, H Y; Chu, J F; Li, P; Li, N; Lv, Z H

2015-01-01

This study was carried out to test expression of transient receptor potential vanilloid1 (TRPV1) in urothelium of female patients with overactive bladder (OAB) and explore clinical significance of TRPV1 in diagnosing female OAB. TRPV1 expression in urothelium of female OAB patients (n=21) and healthy females (n=9) was detected using Strept Avidin-Biotin Complex (SABC), an immunohistochemical method and image analysis system. Relative content of TRPV1 was expressed by average optical density (AOD) and was analyzed through data of urodynamics. Compared to TRPV1 expression in urothelium of healthy females (AOD 0.3658 ± 0.1009), TRPV1 expression in OAB patients was much higher (AOD 0.4834 ± 0.1252) and the difference was significant P less than 0.05. Observation and comparison in clinic of urodynamic parameters of female patients and healthy females revealed that the former had lower indexes with remarkable differences (P less than 0.05) such as Qmax, first desire volume (FDV), strong desire volume (SDV), maximum cyst capacity (MCC) and bladder compliance (BC). Thus high expression of TRPV1 in urothelium of female OAB patients is closely correlated to OAB occurrence, showing great importance of improved bladder sensitivity in female OAB occurrence mechanism.

MOLECULAR AND MORPHOLOGICAL CHANGES IN ZEBRAFISH FOLLOWING TRANSIENT ETHANOL EXPOSURE DURING DEFINED DEVELOPMENTAL STAGES

PubMed Central

Zhang, Chengjin; Frazier, Jared M.; Chen, Hao; Liu, Yao; Lee, Ju-Ahng; Cole, Gregory J.

2014-01-01

Alcohol is a teratogen that has diverse effects on brain and craniofacial development, leading to a constellation of developmental disorders referred to as fetal alcohol spectrum disorder (FASD). The molecular basis of ethanol insult remains poorly understood, as does the relationship between molecular and behavioral changes as a consequence of prenatal ethanol exposure. Zebrafish embryos were exposed to a range of ethanol concentrations (0.5–5.0%) during defined developmental stages, and examined for morphological phenotypes characteristic of FASD. Embryos were also analyzed by in situ hybridization for changes in expression of defined cell markers for neural cell types that are sonic hedgehog-dependent. We show that transient binge-like ethanol exposures during defined developmental stages, such as early gastrulation and early neurulation, result in a range of phenotypes and changes in expression of Shh-dependent genes. The severity of fetal alcohol syndrome (FAS) morphological phenotypes, such as microphthalmia, depends on the embryonic stage and concentration of alcohol exposure, as does diminution of retinal Pax6a or forebrain and hindbrain GAD1 gene expression. We also show that changes in eye and brain morphology correlate with changes in Pax6a and GAD1 gene expression. Our results therefore show that transient binge-like ethanol exposures in zebrafish embryos produce the stereotypical morphological phenotypes of FAS, with the severity of phenotypes depending on the developmental stage and alcohol concentration of exposure. PMID:24929233

4-Phenylbutyrate stimulates Hsp70 expression through the Elp2 component of elongator and STAT-3 in cystic fibrosis epithelial cells.

PubMed

Suaud, Laurence; Miller, Katelyn; Panichelli, Ashley E; Randell, Rachel L; Marando, Catherine M; Rubenstein, Ronald C

2011-12-30

Sodium 4-phenylbutyrate (4PBA) corrects trafficking of ΔF508-CFTR in Cystic Fibrosis (CF) epithelia, which is hypothesized to, at least in part, result from increased expression of Hsp70 (stress-induced 70 kDa heat shock protein). To identify other 4PBA-regulated proteins that may promote correction of ΔF508 trafficking, we performed differential display RT-PCR on mRNA from IB3-1 CF bronchiolar epithelial cells treated for 0-24 h with 1 mM 4PBA. In this screen, a STAT-3 (signal transducer and activator of transcription-3)-interacting protein, StIP-1 that regulates STAT-3 activation had transiently increased expression. StIP-1 is identical to Elongator protein 2 (Elp2), a component of the Elongator complex that regulates RNA polymerase II. Previous studies have suggested that Elongator regulates Hsp70 mRNA transcription, and that the Hsp70 promoter contains functional STAT-3-binding sites. We therefore tested the hypothesis that 4PBA increases Hsp70 expression by an Elongator- and STAT-3-dependent mechanism. 4PBA treatment of IB3-1 CF bronchiolar epithelial cells caused transiently increased expression of Hsp70 protein, as well as Elp2 protein and mRNA. Elp2 depletion by transfection of small interfering RNAs, reduced both Elp2 and Hsp70 protein expression. 4PBA also caused transient activation of STAT-3, and increased abundance of nuclear proteins that bind to the STAT-3-responsive element of the Hsp70 promoter. Luciferase reporter assays demonstrated that both Elp2 overexpression and 4PBA increase Hsp70 promoter activity, while Elp2 depletion blocked the ability of 4PBA to stimulate Hsp70 promoter activity. Together, these data suggest that Elp2 and STAT-3 mediate, at least in part, the stimulation of Hsp70 expression by 4PBA.

4-Phenylbutyrate Stimulates Hsp70 Expression through the Elp2 Component of Elongator and STAT-3 in Cystic Fibrosis Epithelial Cells*

PubMed Central

Suaud, Laurence; Miller, Katelyn; Panichelli, Ashley E.; Randell, Rachel L.; Marando, Catherine M.; Rubenstein, Ronald C.

2011-01-01

Sodium 4-phenylbutyrate (4PBA) corrects trafficking of ΔF508-CFTR in Cystic Fibrosis (CF) epithelia, which is hypothesized to, at least in part, result from increased expression of Hsp70 (stress-induced 70 kDa heat shock protein). To identify other 4PBA-regulated proteins that may promote correction of ΔF508 trafficking, we performed differential display RT-PCR on mRNA from IB3-1 CF bronchiolar epithelial cells treated for 0–24 h with 1 mm 4PBA. In this screen, a STAT-3 (signal transducer and activator of transcription-3)-interacting protein, StIP-1 that regulates STAT-3 activation had transiently increased expression. StIP-1 is identical to Elongator protein 2 (Elp2), a component of the Elongator complex that regulates RNA polymerase II. Previous studies have suggested that Elongator regulates Hsp70 mRNA transcription, and that the Hsp70 promoter contains functional STAT-3-binding sites. We therefore tested the hypothesis that 4PBA increases Hsp70 expression by an Elongator- and STAT-3-dependent mechanism. 4PBA treatment of IB3-1 CF bronchiolar epithelial cells caused transiently increased expression of Hsp70 protein, as well as Elp2 protein and mRNA. Elp2 depletion by transfection of small interfering RNAs, reduced both Elp2 and Hsp70 protein expression. 4PBA also caused transient activation of STAT-3, and increased abundance of nuclear proteins that bind to the STAT-3-responsive element of the Hsp70 promoter. Luciferase reporter assays demonstrated that both Elp2 overexpression and 4PBA increase Hsp70 promoter activity, while Elp2 depletion blocked the ability of 4PBA to stimulate Hsp70 promoter activity. Together, these data suggest that Elp2 and STAT-3 mediate, at least in part, the stimulation of Hsp70 expression by 4PBA. PMID:22069317

Helios expression and Foxp3 TSDR methylation of IFNy+ and IFNy- Treg from kidney transplant recipients with good long-term graft function.

PubMed

Trojan, Karina; Unterrainer, Christian; Weimer, Rolf; Bulut, Nuray; Morath, Christian; Aly, Mostafa; Zhu, Li; Opelz, Gerhard; Daniel, Volker

2017-01-01

There is circumstantial evidence that IFNy+ Treg might have clinical relevance in transplantation. IFNy+ Treg express IFNy receptors and are induced by IFNy. In the present study we investigated in kidney transplant recipients with good long-term stable graft function the absolute cell counts of IFNy+ Treg subsets and whether their expression of Foxp3 is stable or transient. Helios expression determined by eight-color-fluorescence flow cytometry and methylation status of the Foxp3 Treg specific demethylation region (TSDR) served as indicators for stability of Foxp3 expression. Methylation status was investigated in enriched IFNy+ and IFNy- Treg preparations originating from peripheral blood using high resolution melt analysis. A total of 136 transplant recipients and 52 healthy controls were studied. Proportions of IFNy+ Treg were similar in patients and healthy controls (0.05% and 0.04% of all CD4+ lymphocytes; p = n.s.). Patients also had similar absolute counts of IFNy producing Helios+ and Helios- Treg (p = n.s.). Most of the IFNy+ and IFNy- Treg in transplant recipients had a methylated Foxp3 TSDR, however, there was a sizeable proportion of IFNy+ and IFNy- Treg with demethylated Foxp3 TSDR. Male and female patients showed more frequently methylated IFNy+ and IFNy- Treg than male and female controls (all p<0.05). Kidney transplant recipients with good long-term stable graft function have similar levels of IFNy+ Treg as healthy controls. IFNy+ and IFNy- Treg subsets in patients consist of cells with stable and cells with transient Foxp3 expression; however, patients showed more frequently methylated IFNy+ and IFNy- Treg than controls. The data show increased levels of Treg subsets with stable as well as transient Foxp3 expression in patients with stable allograft acceptance compared to healthy controls.

Can We Distinguish Emotions from Faces? Investigation of Implicit and Explicit Processes of Peak Facial Expressions.

PubMed

Xiao, Ruiqi; Li, Xianchun; Li, Lin; Wang, Yanmei

2016-01-01

Most previous studies on facial expression recognition have focused on the moderate emotions; to date, few studies have been conducted to investigate the explicit and implicit processes of peak emotions. In the current study, we used transiently peak intense expression images of athletes at the winning or losing point in competition as materials, and investigated the diagnosability of peak facial expressions at both implicit and explicit levels. In Experiment 1, participants were instructed to evaluate isolated faces, isolated bodies, and the face-body compounds, and eye-tracking movement was recorded. The results revealed that the isolated body and face-body congruent images were better recognized than isolated face and face-body incongruent images, indicating that the emotional information conveyed by facial cues was ambiguous, and the body cues influenced facial emotion recognition. Furthermore, eye movement records showed that the participants displayed distinct gaze patterns for the congruent and incongruent compounds. In Experiment 2A, the subliminal affective priming task was used, with faces as primes and bodies as targets, to investigate the unconscious emotion perception of peak facial expressions. The results showed that winning face prime facilitated reaction to winning body target, whereas losing face prime inhibited reaction to winning body target, suggesting that peak facial expressions could be perceived at the implicit level. In general, the results indicate that peak facial expressions cannot be consciously recognized but can be perceived at the unconscious level. In Experiment 2B, revised subliminal affective priming task and a strict awareness test were used to examine the validity of unconscious perception of peak facial expressions found in Experiment 2A. Results of Experiment 2B showed that reaction time to both winning body targets and losing body targets was influenced by the invisibly peak facial expression primes, which indicated the unconscious perception of peak facial expressions.

Can We Distinguish Emotions from Faces? Investigation of Implicit and Explicit Processes of Peak Facial Expressions

PubMed Central

Xiao, Ruiqi; Li, Xianchun; Li, Lin; Wang, Yanmei

2016-01-01

Most previous studies on facial expression recognition have focused on the moderate emotions; to date, few studies have been conducted to investigate the explicit and implicit processes of peak emotions. In the current study, we used transiently peak intense expression images of athletes at the winning or losing point in competition as materials, and investigated the diagnosability of peak facial expressions at both implicit and explicit levels. In Experiment 1, participants were instructed to evaluate isolated faces, isolated bodies, and the face-body compounds, and eye-tracking movement was recorded. The results revealed that the isolated body and face-body congruent images were better recognized than isolated face and face-body incongruent images, indicating that the emotional information conveyed by facial cues was ambiguous, and the body cues influenced facial emotion recognition. Furthermore, eye movement records showed that the participants displayed distinct gaze patterns for the congruent and incongruent compounds. In Experiment 2A, the subliminal affective priming task was used, with faces as primes and bodies as targets, to investigate the unconscious emotion perception of peak facial expressions. The results showed that winning face prime facilitated reaction to winning body target, whereas losing face prime inhibited reaction to winning body target, suggesting that peak facial expressions could be perceived at the implicit level. In general, the results indicate that peak facial expressions cannot be consciously recognized but can be perceived at the unconscious level. In Experiment 2B, revised subliminal affective priming task and a strict awareness test were used to examine the validity of unconscious perception of peak facial expressions found in Experiment 2A. Results of Experiment 2B showed that reaction time to both winning body targets and losing body targets was influenced by the invisibly peak facial expression primes, which indicated the unconscious perception of peak facial expressions. PMID:27630604

Identification and Characterization of (3Z):(2E)-Hexenal Isomerases from Cucumber

PubMed Central

Spyropoulou, Eleni A.; Dekker, Henk L.; Steemers, Luuk; van Maarseveen, Jan H.; de Koster, Chris G.; Haring, Michel A.; Schuurink, Robert C.; Allmann, Silke

2017-01-01

E-2-hexenal is a volatile compound that is commonly emitted by wounded or stressed plants. It belongs to the group of so-called green leaf volatiles (GLVs), which play an important role in transferring information to plants and insects. While most biosynthetic enzymes upstream of E-2-hexenal have been studied extensively, much less is known about the enzyme responsible for the conversion from Z-3- to E-2-hexenal. In this study we have identified two (3Z):(2E)-hexenal isomerases (HIs) from cucumber fruits by classical biochemical fractionation techniques and we were able to confirm their activity by heterologous expression. Recombinant protein of the HIs did not only convert the leaf aldehyde Z-3-hexenal to E-2-hexenal, but also (Z,Z)-3,6-nonadienal to (E,Z)-2,6-nonadienal, these last two representing major flavor volatiles of cucumber fruits. Transient expression of the cucumber HIs in Nicotiana benthamiana leaves drastically changed the GLV bouquet of damaged plants from a Z-3- to an E-2-enriched GLV profile. Furthermore, transcriptional analysis revealed that the two HIs showed distinct expression patterns. While HI-1 was specifically expressed in the flesh of cucumber fruits HI-2 was expressed in leaves as well. Interestingly, wounding of cucumber leaves caused only a slight increase in HI-2 transcript levels. These results demonstrate that cucumber HIs are responsible for the rearrangement of Z-3-aldehydes in both leaves and fruits. Future research will reveal the physiological importance of an increased conversion to E-2-aldehydes for plants and insects. PMID:28824678

Identification and Characterization of (3Z):(2E)-Hexenal Isomerases from Cucumber.

PubMed

Spyropoulou, Eleni A; Dekker, Henk L; Steemers, Luuk; van Maarseveen, Jan H; de Koster, Chris G; Haring, Michel A; Schuurink, Robert C; Allmann, Silke

2017-01-01

E -2-hexenal is a volatile compound that is commonly emitted by wounded or stressed plants. It belongs to the group of so-called green leaf volatiles (GLVs), which play an important role in transferring information to plants and insects. While most biosynthetic enzymes upstream of E -2-hexenal have been studied extensively, much less is known about the enzyme responsible for the conversion from Z -3- to E -2-hexenal. In this study we have identified two (3 Z ):(2 E )-hexenal isomerases (HIs) from cucumber fruits by classical biochemical fractionation techniques and we were able to confirm their activity by heterologous expression. Recombinant protein of the HIs did not only convert the leaf aldehyde Z -3-hexenal to E -2-hexenal, but also ( Z,Z )-3,6-nonadienal to ( E,Z )-2,6-nonadienal, these last two representing major flavor volatiles of cucumber fruits. Transient expression of the cucumber HIs in Nicotiana benthamiana leaves drastically changed the GLV bouquet of damaged plants from a Z -3- to an E -2-enriched GLV profile. Furthermore, transcriptional analysis revealed that the two HIs showed distinct expression patterns. While HI-1 was specifically expressed in the flesh of cucumber fruits HI-2 was expressed in leaves as well. Interestingly, wounding of cucumber leaves caused only a slight increase in HI-2 transcript levels. These results demonstrate that cucumber HIs are responsible for the rearrangement of Z -3-aldehydes in both leaves and fruits. Future research will reveal the physiological importance of an increased conversion to E -2-aldehydes for plants and insects.

Control of early cardiac-specific transcription of Nkx2-5 by a GATA-dependent enhancer.

PubMed

Lien, C L; Wu, C; Mercer, B; Webb, R; Richardson, J A; Olson, E N

1999-01-01

The homeobox gene Nkx2-5 is the earliest known marker of the cardiac lineage in vertebrate embryos. Nkx2-5 expression is first detected in mesodermal cells specified to form heart at embryonic day 7.5 in the mouse and expression is maintained throughout the developing and adult heart. In addition to the heart, Nkx2-5 is transiently expressed in the developing pharynx, thyroid and stomach. To investigate the mechanisms that initiate cardiac transcription during embryogenesis, we analyzed the Nkx2-5 upstream region for regulatory elements sufficient to direct expression of a lacZ transgene in the developing heart of transgenic mice. We describe a cardiac enhancer, located about 9 kilobases upstream of the Nkx2-5 gene, that fully recapitulates the expression pattern of the endogenous gene in cardiogenic precursor cells from the onset of cardiac lineage specification and throughout the linear and looping heart tube. Thereafter, as the atrial and ventricular chambers become demarcated, enhancer activity becomes restricted to the developing right ventricle. Transcription of Nkx2-5 in pharynx, thyroid and stomach is controlled by regulatory elements separable from the cardiac enhancer. This distal cardiac enhancer contains a high-affinity binding site for the cardiac-restricted zinc finger transcription factor GATA4 that is essential for transcriptional activity. These results reveal a novel GATA-dependent mechanism for activation of Nkx2-5 transcription in the developing heart and indicate that regulation of Nkx2-5 is controlled in a modular manner, with multiple regulatory regions responding to distinct transcriptional networks in different compartments of the developing heart.

The orphan receptor Rev-erbα gene is a target of the circadian clock pacemaker

PubMed Central

Triqueneaux, Gérard; Thenot, Sandrine; Kakizawa, Tomoko; Antoch, Marina P; Safi, Rachid; Takahashi, Joseph S; Delaunay, Franck; Laudet, Vincent

2013-01-01

Rev-erbα is a ubiquitously expressed orphan nuclear receptor which functions as a constitutive transcriptional repressor and is expressed in vertebrates according to a robust circadian rhythm. We report here that two Rev-erbα mRNA isoforms, namely Rev-erbα1 and Rev-erbα2, are generated through alternative promoter usage and that both show a circadian expression pattern in an in vitro system using serum-shocked fibroblasts. Both promoter regions P1 (Rev-erbα1) and P2 (Rev-erbα2) contain several E-box DNA sequences, which function as response elements for the core circadian-clock components: CLOCK and BMAL1. The CLOCK–BMAL1 heterodimer stimulates the activity of both P1 and P2 promoters in transient transfection assay by 3–6-fold. This activation was inhibited by the overexpression of CRY1, a component of the negative limb of the circadian transcriptional loop. Critical E-box elements were mapped within both promoters. This regulation is conserved in vertebrates since we found that the CLOCK–BMAL1 heterodimer also regulates the zebrafish Rev-erbα gene. In line with these data Rev-erbα circadian expression was strongly impaired in the livers of Clock mutant mice and in the pineal glands of zebrafish embryos treated with Clock and Bmal1 antisense oligonucleotides. Together these data demonstrate that CLOCK is a critical regulator of Rev-erbα circadian gene expression in evolutionarily distant vertebrates and suggest a role for Rev-erbα in the circadian clock output. PMID:15591021

Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

NASA Technical Reports Server (NTRS)

Poovaiah, B. W.; Xia, M.; Liu, Z.; Wang, W.; Yang, T.; Sathyanarayanan, P. V.; Franceschi, V. R.

1999-01-01

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

Alk5/Runx1 signaling mediated by extracellular vesicles promotes vascular repair in acute respiratory distress syndrome.

PubMed

Shah, Trushil; Qin, Shanshan; Vashi, Mona; Predescu, Dan N; Jeganathan, Niranjan; Bardita, Cristina; Ganesh, Balaji; diBartolo, Salvatore; Fogg, Louis F; Balk, Robert A; Predescu, Sanda A

2018-06-22

Pulmonary endothelial cells' (ECs) injury and apoptotic death are necessary and sufficient for the pathogenesis of the acute respiratory distress syndrome (ARDS), regardless of epithelial damage. Interaction of dysfunctional ECs with circulatory extracellular vesicles (EVs) holds therapeutic promise in ARDS. However, the presence in the blood of long-term ARDS survivors of EVs with a distinct phenotype compared to the EVs of non-surviving patients is not reported. With a multidisciplinary translational approach, we studied EVs from the blood of 33 patients with moderate-to-severe ARDS. The EVs were isolated from the blood of ARDS and control subjects. Immunoblotting and magnetic beads immunoisolation complemented by standardized flow cytometry and nanoparticles tracking analyses identified in the ARDS patients a subset of EVs with mesenchymal stem cell (MSC) origin (CD73 + CD105 + Cd34 - CD45 - ). These EVs have 4.7-fold greater counts compared to controls and comprise the transforming growth factor-beta receptor I (TβRI)/Alk5 and the Runx1 transcription factor. Time course analyses showed that the expression pattern of two Runx1 isoforms is critical for ARDS outcome: the p52 isoform shows a continuous expression, while the p66 is short-lived. A high ratio Runx1p66/p52 provided a survival advantage, regardless of age, sex, disease severity or length of stay in the intensive care unit. Moreover, the Runx1p66 isoform is transiently expressed by cultured human bone marrow-derived MSCs, it is released in the EVs recoverable from the conditioned media and stimulates the proliferation of lipopolysaccharide (LPS)-treated ECs. The findings are consistent with a causal effect of Runx1p66 expression on EC proliferation. Furthermore, morphological and functional assays showed that the EVs bearing the Runx1p66 enhanced junctional integrity of LPS-injured ECs and decreased lung histological severity in the LPS-treated mice. The expression pattern of Runx1 isoforms might be a reliable circulatory biomarker of ARDS activity and a novel determinant of the molecular mechanism for lung vascular/tissue repair and recovery after severe injury.

Transient and permanent changes in DNA methylation patterns in inorganic arsenic-mediated epithelial-to-mesenchymal transition.

PubMed

Eckstein, Meredith; Rea, Matthew; Fondufe-Mittendorf, Yvonne N

2017-09-15

Chronic low dose inorganic arsenic exposure causes cells to take on an epithelial-to-mesenchymal phenotype, which is a crucial process in carcinogenesis. Inorganic arsenic is not a mutagen and thus epigenetic alterations have been implicated in this process. Indeed, during the epithelial-to-mesenchymal transition, morphologic changes to cells correlate with changes in chromatin structure and gene expression, ultimately driving this process. However, studies on the effects of inorganic arsenic exposure/withdrawal on the epithelial-to-mesenchymal transition and the impact of epigenetic alterations in this process are limited. In this study we used high-resolution microarray analysis to measure the changes in DNA methylation in cells undergoing inorganic arsenic-induced epithelial-to-mesenchymal transition, and on the reversal of this process, after removal of the inorganic arsenic exposure. We found that cells exposed to chronic, low-dose inorganic arsenic exposure showed 30,530 sites were differentially methylated, and with inorganic arsenic withdrawal several differential methylated sites were reversed, albeit not completely. Furthermore, these changes in DNA methylation mainly correlated with changes in gene expression at most sites tested but not at all. This study suggests that DNA methylation changes on gene expression are not clear-cut and provide a platform to begin to uncover the relationship between DNA methylation and gene expression, specifically within the context of inorganic arsenic treatment. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Anthocyanin biosynthesis regulation of DhMYB2 and DhbHLH1 in Dendrobium hybrids petals.

PubMed

Li, Chonghui; Qiu, Jian; Ding, Ling; Huang, Mingzhong; Huang, Surong; Yang, Guangsui; Yin, Junmei

2017-03-01

Dendrobium hybrids orchid are popular throughout the world. They have various floral color and pigmentation patterns that are mainly caused by anthocyanins. It is well established that anthocyanin biosynthesis is regulated by the interplay between MYB and bHLH transcription factors (TF) in most plants. In this study, we identified one R2R3-MYB gene, DhMYB2, and one bHLH gene, DhbHLH1, from a Dendrobium hybrid. Their expression profiles were related to anthocyanin pigmentation in Dendrobium petals. Transient over-expression of these two TF genes showed that both DhMYB2 and DhbHLH1 resulted in anthocyanin production in white petals. The interaction between the two TFs was observed in vitro. In different Dendrobium hybrids petals with various pigmentations, DhMYB2 and DhbHLH1 were co-expressed with DhDFR and DhANS, which are regarded as potential regulatory targets of the two TFs. In flowers with distinct purple lips but white or yellow petals/sepals, the expression of DhbHLH1 was only related to anthocyanin accumulation in the lips. Taken together, DhMYB2 interacted with DhbHLH1 to regulate anthocyanin production in Dendrobium hybrid petals. DhbHLH1 was also responsible for the distinct anthocyanin pigmentation in lip tissues. The functional characterization of DhMYB2 and DhbHLH1 will improve understanding of anthocyanin biosynthesis modulation in Dendrobium orchids. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

An expert protocol for immunofluorescent detection of calcium channels in tsA-201 cells.

PubMed

Koch, Peter; Herzig, Stefan; Matthes, Jan

Pore-forming subunits of voltage gated calcium channels (VGCC) are large membrane proteins (260kDa) containing 24 transmembrane domains. Despite transfection with viral promoter driven vectors, biochemical analysis of VGCC is often hampered by rather low expression levels in heterologous systems rendering VGCC challenging targets. Especially in immunofluorescent detection, calcium channels are demanding proteins. We provide an expert step-by-step protocol with adapted conditions for handling procedures (tsA-201 cell culture, transient transfection, incubation time and temperature at 28°C or 37°C and immunostaining) to address the L-type calcium-channel pore Ca v 1.2 in an immunofluorescent approach. We performed immunocytochemical analysis of Ca v 1.2 expression at single-cell level in combination with detection of different markers for cellular organelles. We show confluency levels and shapes of tsA-201 cells at different time points during an experiment. Our experiments reveal sufficient levels of Ca v 1.2 protein and a correct Ca v 1.2 expression pattern in polygonal shaped cells already 12h after transfection. A sequence of elaborated protocol modifications allows subcellular localization analysis of Ca v 1.2 in an immunocytochemical approach. We provide a protocol that may be used to achieve insights into physiological and pathophysiological processes involving voltage gated calcium channels. Our protocol may be used for expression analysis of other challenging proteins and efficient overexpression may be exploited in related biochemical techniques requiring immunolabels. Copyright © 2016 Elsevier Inc. All rights reserved.

The rice YABBY1 gene is involved in the feedback regulation of gibberellin metabolism.

PubMed

Dai, Mingqiu; Zhao, Yu; Ma, Qian; Hu, Yongfeng; Hedden, Peter; Zhang, Qifa; Zhou, Dao-Xiu

2007-05-01

Gibberellin (GA) biosynthesis is regulated by feedback control providing a mechanism for GA homeostasis in plants. However, regulatory elements involved in the feedback control are not known. In this report, we show that a rice (Oryza sativa) YABBY1 (YAB1) gene had a similar expression pattern as key rice GA biosynthetic genes GA3ox2 and GA20ox2. Overexpression of YAB1 in transgenic rice resulted in a semidwarf phenotype that could be fully rescued by applied GA. Quantification of the endogenous GA content revealed increases of GA(20) and decreases of GA(1) levels in the overexpression plants, in which the transcripts of the biosynthetic gene GA3ox2 were decreased. Cosuppression of YAB1 in transgenic plants induced expression of GA3ox2. The repression of GA3ox2 could be obtained upon treatment by dexamethasone of transgenic plants expressing a YAB1-glucocorticoid receptor fusion. Importantly, we show that YAB1 bound to a GA-responsive element within the GA3ox2 promoter. In addition, the expression of YAB1 was deregulated in GA biosynthesis and signaling mutants and could be either transiently induced by GA or repressed by a GA inhibitor. Finally, either overexpression or cosuppression of YAB1 impaired GA-mediated repression of GA3ox2. These data together suggest that YAB1 is involved in the feedback regulation of GA biosynthesis in rice.

A visual study of radial inward choked flow of liquid nitrogen.

NASA Technical Reports Server (NTRS)

Hendricks, R. C.; Simoneau, R. J.; Hsu, Y. Y.

1973-01-01

Data and high speed movies were acquired on pressurized subcooled liquid nitrogen flowing radially inward through a 0.0076 cm gap. The stagnation pressure ranged from 0.7 to 4 MN/sq m. Steady radial inward choked flow appears equivalent to steady choked flow through axisymmetric nozzles. Transient choked flows through the radial gap are not uniform and the discharge pattern appears as nonuniform impinging jets. The critical mass flow rate data for the transient case appear different from those for the steady case. On the mass flow rate vs pressure map, the slope and separation of the isotherms appear to be less for transient than for steady radial choked flow.

Cortical cholinergic signaling controls the detection of cues

PubMed Central

Gritton, Howard J.; Howe, William M.; Mallory, Caitlin S.; Hetrick, Vaughn L.; Berke, Joshua D.; Sarter, Martin

2016-01-01

The cortical cholinergic input system has been described as a neuromodulator system that influences broadly defined behavioral and brain states. The discovery of phasic, trial-based increases in extracellular choline (transients), resulting from the hydrolysis of newly released acetylcholine (ACh), in the cortex of animals reporting the presence of cues suggests that ACh may have a more specialized role in cognitive processes. Here we expressed channelrhodopsin or halorhodopsin in basal forebrain cholinergic neurons of mice with optic fibers directed into this region and prefrontal cortex. Cholinergic transients, evoked in accordance with photostimulation parameters determined in vivo, were generated in mice performing a task necessitating the reporting of cue and noncue events. Generating cholinergic transients in conjunction with cues enhanced cue detection rates. Moreover, generating transients in noncued trials, where cholinergic transients normally are not observed, increased the number of invalid claims for cues. Enhancing hits and generating false alarms both scaled with stimulation intensity. Suppression of endogenous cholinergic activity during cued trials reduced hit rates. Cholinergic transients may be essential for synchronizing cortical neuronal output driven by salient cues and executing cue-guided responses. PMID:26787867

Dimensionless figure of merit and its efficiency estimation for transient response of thermoelectric module based on impedance spectroscopy

NASA Astrophysics Data System (ADS)

Otsuka, Mioko; Hasegawa, Yasuhiro; Arisaka, Taichi; Shinozaki, Ryo; Morita, Hiroyuki

2017-11-01

The dimensionless figure of merit and its efficiency for the transient response of a Π-shaped thermoelectric module are estimated according to the theory of impedance spectroscopy. The effective dimensionless figure of merit is described as a function of the product of the characteristic time to reduce the temperature and the representative angular frequency of the module, which is expressed by the thermal diffusivity and the length of the elements used. The characteristic time required for achieving a higher dimensionless figure of merit and efficiency is derived quantitatively for the transient response using the properties of a commercial thermoelectric module.

Defects and spatiotemporal disorder in a pattern of falling liquid columns

NASA Astrophysics Data System (ADS)

Brunet, Philippe; Limat, Laurent

2004-10-01

Disordered regimes of a one-dimensional pattern of liquid columns hanging below an overflowing circular dish are investigated experimentally. The interaction of two basic dynamical modes (oscillations and drift) combined with the occurrence of defects (birth of new columns, disappearances by coalescences of two columns) leads to spatiotemporal chaos. When the flow rate is progressively increased, a continuous transition between transient and permanent chaos is pointed into evidence. We introduce the rate of defects as the sole relevant quantity to quantify this “turbulence” without ambiguity. Statistics on both transient and endlessly chaotic regimes enable to define a critical flow rate around which exponents are extracted. Comparisons are drawn with other interfacial pattern-forming systems, where transition towards chaos follows similar steps. Qualitatively, careful examinations of the global dynamics show that the contamination processes are nonlocal and involve the propagation of blocks of elementary laminar states (such as propagative domains or local oscillations), emitted near the defects, which turn out to be essential ingredients of this self-sustained disorder.

Transient anhedonia phenotype and altered circadian timing of behaviour during night-time dim light exposure in Per3-/- mice, but not wildtype mice.

PubMed

Martynhak, Bruno Jacson; Hogben, Alexandra L; Zanos, Panos; Georgiou, Polymnia; Andreatini, Roberto; Kitchen, Ian; Archer, Simon N; von Schantz, Malcolm; Bailey, Alexis; van der Veen, Daan R

2017-01-10

Industrialisation greatly increased human night-time exposure to artificial light, which in animal models is a known cause of depressive phenotypes. Whilst many of these phenotypes are 'direct' effects of light on affect, an 'indirect' pathway via altered sleep-wake timing has been suggested. We have previously shown that the Period3 gene, which forms part of the biological clock, is associated with altered sleep-wake patterns in response to light. Here, we show that both wild-type and Per3 -/- mice showed elevated levels of circulating corticosterone and increased hippocampal Bdnf expression after 3 weeks of exposure to dim light at night, but only mice deficient for the PERIOD3 protein (Per3 -/- ) exhibited a transient anhedonia-like phenotype, observed as reduced sucrose preference, in weeks 2-3 of dim light at night, whereas WT mice did not. Per3 -/- mice also exhibited a significantly smaller delay in behavioural timing than WT mice during weeks 1, 2 and 4 of dim light at night exposure. When treated with imipramine, neither Per3 -/- nor WT mice exhibited an anhedonia-like phenotype, and neither genotypes exhibited a delay in behavioural timing in responses to dLAN. While the association between both Per3 -/- phenotypes remains unclear, both are alleviated by imipramine treatment during dim night-time light.

In situ hybridisation of a large repertoire of muscle-specific transcripts in fish larvae: the new superficial slow-twitch fibres exhibit characteristics of fast-twitch differentiation.

PubMed

Chauvigné, F; Ralliere, C; Cauty, C; Rescan, P Y

2006-01-01

Much of the present information on muscle differentiation in fish concerns the early embryonic stages. To learn more about the maturation and the diversification of the fish myotomal fibres in later stages of ontogeny, we investigated, by means of in situ hybridisation, the developmental expression of a large repertoire of muscle-specific genes in trout larvae from hatching to yolk resorption. At hatching, transcripts for fast and slow muscle protein isoforms, namely myosins, tropomyosins, troponins and myosin binding protein C were present in the deep fast and the superficial slow areas of the myotome, respectively. During myotome expansion that follows hatching, the expression of fast isoforms became progressively confined to the borders of the fast muscle mass, whereas, in contrast, slow muscle isoform transcripts were uniformly expressed in all the slow fibres. Transcripts for several enzymes involved in oxidative metabolism such as citrate synthase, cytochrome oxidase component IV and succinate dehydrogenase, were present throughout the whole myotome of hatching embryos but in later stages became concentrated in slow fibre as well as in lateral fast fibres. Surprisingly, the slow fibres that are added externally to the single superficial layer of the embryonic (original) slow muscle fibres expressed not only slow twitch muscle isoforms but also, transiently, a subset of fast twitch muscle isoforms including MyLC1, MyLC3, MyHC and myosin binding protein C. Taken together these observations show that the growth of the myotome of the fish larvae is associated with complex patterns of muscular gene expression and demonstrate the unexpected presence of fast muscle isoform-expressing fibres in the most superficial part of the slow muscle.

By Different Cellular Mechanisms, Lymphatic Vessels Sprout by Endothelial Cell Recruitment Whereas Blood Vessels Grow by Vascular Expansion

NASA Technical Reports Server (NTRS)

Parsons-Wingerter, Patricia; McKay, Terri L.; Leontiev, Dmitry; Condrich, Terence K.; DiCorleto, Paul E.

2005-01-01

The development of effective vascular therapies requires the understanding of all modes of vessel formation contributing to vasculogenesis, angiogenesis (here termed hemangiogenesis) and lymphangiogenesis. We show that lymphangiogenesis proceeds by blind-ended vessel sprouting via recruitment of isolated endothelial progenitor cells to the tips of growing vessels, whereas hemangiogenesis occurs by non-sprouting vessel expansion from the capillary network, during middevelopment in the quail chorioallantoic membrane (CAM). Blood vessels expanded out of capillaries that displayed transient expression of alpha smooth muscle actin (alphaSMA), accompanied by mural recruitment of migratory progenitor cells expressing SMA. Lymphatics and blood vessels were identified by confocal/fluorescence microscopy of vascular endothelial growth factor (VEGF) receptors VEGFR-1 and VEGFR-2, alphaSMA (expressed on CAM blood vessels but not on lymphatics), homeobox transcription factor Prox-1 (specific to CAM lymphatic endothelium), and the quail hematopoetic/vascular marker, QH-1. Expression of VEGFR-1 was highly restricted to blood vessels (primarily capillaries). VEGFR-2 was expressed intensely in isolated hematopoietic cells, lymphatic vessels and moderately in blood vessels. Prox-1 was absent from endothelial progenitor cells prior to lymphatic recruitment. Although vascular endothelial growth factor-165 (VEGF(sub 165)) is a key regulator of numerous cellular processes in hemangiogenesis and vasculogenesis, the role of VEGF(sub 165) in lymphangiogenesis is less clear. Exogenous VEGF(sub 165) increased blood vessel density without changing endogenous modes of vascular/lymphatic vessel formation or marker expression patterns. However, VEGF(sub 165) did increase the frequency of blood vascular anastomoses and strongly induced the antimaturational dissociation of lymphatics from blood vessels, with frequent formation of homogeneous lymphatic networks.

Carbohydrate utilization and the lager yeast transcriptome during brewery fermentation.

PubMed

Gibson, Brian R; Boulton, Chris A; Box, Wendy G; Graham, Neil S; Lawrence, Stephen J; Linforth, Robert S T; Smart, Katherine A

2008-08-01

The fermentable carbohydrate composition of wort and the manner in which it is utilized by yeast during brewery fermentation have a direct influence on fermentation efficiency and quality of the final product. In this study the response of a brewing yeast strain to changes in wort fermentable carbohydrate concentration and composition during full-scale (3275 hl) brewery fermentation was investigated by measuring transcriptome changes with the aid of oligonucleotide-based DNA arrays. Up to 74% of the detectable genes showed a significant (p

The insulin and islet amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical beta-cell nuclear complexes.

PubMed Central

German, M S; Moss, L G; Wang, J; Rutter, W J

1992-01-01

The pancreatic beta cell makes several unique gene products, including insulin, islet amyloid polypeptide (IAPP), and beta-cell-specific glucokinase (beta GK). The functions of isolated portions of the insulin, IAPP, and beta GK promoters were studied by using transient expression and DNA binding assays. A short portion (-247 to -197 bp) of the rat insulin I gene, the FF minienhancer, contains three interacting transcriptional regulatory elements. The FF minienhancer binds at least two nuclear complexes with limited tissue distribution. Sequences similar to that of the FF minienhancer are present in the 5' flanking DNA of the human IAPP and rat beta GK genes and also the rat insulin II and mouse insulin I and II genes. Similar minienhancer constructs from the insulin and IAPP genes function as cell-specific transcriptional regulatory elements and compete for binding of the same nuclear factors, while the beta GK construct competes for protein binding but functions poorly as a minienhancer. These observations suggest that the patterns of expression of the beta-cell-specific genes result in part from sharing the same transcriptional regulators. Images PMID:1549125

Nanog Fluctuations in Embryonic Stem Cells Highlight the Problem of Measurement in Cell Biology.

PubMed

Smith, Rosanna C G; Stumpf, Patrick S; Ridden, Sonya J; Sim, Aaron; Filippi, Sarah; Harrington, Heather A; MacArthur, Ben D

2017-06-20

A number of important pluripotency regulators, including the transcription factor Nanog, are observed to fluctuate stochastically in individual embryonic stem cells. By transiently priming cells for commitment to different lineages, these fluctuations are thought to be important to the maintenance of, and exit from, pluripotency. However, because temporal changes in intracellular protein abundances cannot be measured directly in live cells, fluctuations are typically assessed using genetically engineered reporter cell lines that produce a fluorescent signal as a proxy for protein expression. Here, using a combination of mathematical modeling and experiment, we show that there are unforeseen ways in which widely used reporter strategies can systematically disturb the dynamics they are intended to monitor, sometimes giving profoundly misleading results. In the case of Nanog, we show how genetic reporters can compromise the behavior of important pluripotency-sustaining positive feedback loops, and induce a bifurcation in the underlying dynamics that gives rise to heterogeneous Nanog expression patterns in reporter cell lines that are not representative of the wild-type. These findings help explain the range of published observations of Nanog variability and highlight the problem of measurement in live cells. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells.

PubMed

Engbers, Jordan D T; Anderson, Dustin; Asmara, Hadhimulya; Rehak, Renata; Mehaffey, W Hamish; Hameed, Shahid; McKay, Bruce E; Kruskic, Mirna; Zamponi, Gerald W; Turner, Ray W

2012-02-14

Encoding sensory input requires the expression of postsynaptic ion channels to transform key features of afferent input to an appropriate pattern of spike output. Although Ca(2+)-activated K(+) channels are known to control spike frequency in central neurons, Ca(2+)-activated K(+) channels of intermediate conductance (KCa3.1) are believed to be restricted to peripheral neurons. We now report that cerebellar Purkinje cells express KCa3.1 channels, as evidenced through single-cell RT-PCR, immunocytochemistry, pharmacology, and single-channel recordings. Furthermore, KCa3.1 channels coimmunoprecipitate and interact with low voltage-activated Cav3.2 Ca(2+) channels at the nanodomain level to support a previously undescribed transient voltage- and Ca(2+)-dependent current. As a result, subthreshold parallel fiber excitatory postsynaptic potentials (EPSPs) activate Cav3 Ca(2+) influx to trigger a KCa3.1-mediated regulation of the EPSP and subsequent after-hyperpolarization. The Cav3-KCa3.1 complex provides powerful control over temporal summation of EPSPs, effectively suppressing low frequencies of parallel fiber input. KCa3.1 channels thus contribute to a high-pass filter that allows Purkinje cells to respond preferentially to high-frequency parallel fiber bursts characteristic of sensory input.

Aging and CaMKII alter intracellular Ca2+ transients and heart rhythm in Drosophila melanogaster.

PubMed

Santalla, Manuela; Valverde, Carlos A; Harnichar, Ezequiel; Lacunza, Ezequiel; Aguilar-Fuentes, Javier; Mattiazzi, Alicia; Ferrero, Paola

2014-01-01

Aging is associated to disrupted contractility and rhythmicity, among other cardiovascular alterations. Drosophila melanogaster shows a pattern of aging similar to human beings and recapitulates the arrhythmogenic conditions found in the human heart. Moreover, the kinase CaMKII has been characterized as an important regulator of heart function and an arrhythmogenic molecule that participate in Ca2+ handling. Using a genetically engineered expressed Ca2+ indicator, we report changes in cardiac Ca2+ handling at two different ages. Aging prolonged relaxation, reduced spontaneous heart rate (HR) and increased the occurrence of arrhythmias, ectopic beats and asystoles. Alignment between Drosophila melanogaster and human CaMKII showed a high degree of conservation and indicates that relevant phosphorylation sites in humans are also present in the fruit fly. Inhibition of CaMKII by KN-93 (CaMKII-specific inhibitor), reduced HR without significant changes in other parameters. By contrast, overexpression of CaMKII increased HR and reduced arrhythmias. Moreover, it increased fluorescence amplitude, maximal rate of rise of fluorescence and reduced time to peak fluorescence. These results suggest that CaMKII in Drosophila melanogaster acts directly on heart function and that increasing CaMKII expression levels could be beneficial to improve contractility.

A Flavonoid Compound Promotes Neuronal Differentiation of Embryonic Stem Cells via PPAR-β Modulating Mitochondrial Energy Metabolism.

PubMed

Mei, Yu-Qin; Pan, Zong-Fu; Chen, Wen-Teng; Xu, Min-Hua; Zhu, Dan-Yan; Yu, Yong-Ping; Lou, Yi-Jia

2016-01-01

Relatively little is known regarding mitochondrial metabolism in neuronal differentiation of embryonic stem (ES) cells. By using a small molecule, present research has investigated the pattern of cellular energy metabolism in neural progenitor cells derived from mouse ES cells. Flavonoid compound 4a faithfully facilitated ES cells to differentiate into neurons morphologically and functionally. The expression and localization of peroxisome proliferator-activated receptors (PPARs) were examined in neural progenitor cells. PPAR-β expression showed robust upregulation compared to solvent control. Treatment with PPAR-β agonist L165041 alone or together with compound 4a significantly promoted neuronal differentiation, while antagonist GSK0660 blocked the neurogenesis-promoting effect of compound 4a. Consistently, knockdown of PPAR-β in ES cells abolished compound 4a-induced neuronal differentiation. Interestingly, we found that mitochondrial fusion protein Mfn2 was also abolished by sh-PPAR-β, resulting in abnormal mitochondrial Ca2+ ([Ca2+]M) transients as well as impaired mitochondrial bioenergetics. In conclusion, we demonstrated that by modulating mitochondrial energy metabolism through Mfn2 and mitochondrial Ca2+, PPAR-β took an important role in neuronal differentiation induced by flavonoid compound 4a.

A Flavonoid Compound Promotes Neuronal Differentiation of Embryonic Stem Cells via PPAR-β Modulating Mitochondrial Energy Metabolism

PubMed Central

Mei, Yu-qin; Pan, Zong-fu; Chen, Wen-teng; Xu, Min-hua; Zhu, Dan-yan; Yu, Yong-ping; Lou, Yi-jia

2016-01-01

Relatively little is known regarding mitochondrial metabolism in neuronal differentiation of embryonic stem (ES) cells. By using a small molecule, present research has investigated the pattern of cellular energy metabolism in neural progenitor cells derived from mouse ES cells. Flavonoid compound 4a faithfully facilitated ES cells to differentiate into neurons morphologically and functionally. The expression and localization of peroxisome proliferator-activated receptors (PPARs) were examined in neural progenitor cells. PPAR-β expression showed robust upregulation compared to solvent control. Treatment with PPAR-β agonist L165041 alone or together with compound 4a significantly promoted neuronal differentiation, while antagonist GSK0660 blocked the neurogenesis-promoting effect of compound 4a. Consistently, knockdown of PPAR-β in ES cells abolished compound 4a-induced neuronal differentiation. Interestingly, we found that mitochondrial fusion protein Mfn2 was also abolished by sh-PPAR-β, resulting in abnormal mitochondrial Ca2+ ([Ca2+]M) transients as well as impaired mitochondrial bioenergetics. In conclusion, we demonstrated that by modulating mitochondrial energy metabolism through Mfn2 and mitochondrial Ca2+, PPAR-β took an important role in neuronal differentiation induced by flavonoid compound 4a. PMID:27315062

Distinct activation of tumor necrosis factor-α and interleukin-6 in the spinal cord after surgical incision in rats.

PubMed

Zhang, Yan-Ling; Xu, Jun-Mei; Zhou, Pei; Zhong, Xiao-Lin; Dai, Ru-Ping

2012-06-01

In a previous study, we showed that a deep thoracic incision induces the segmental upregulation of interleukin-1β (IL-1β) in the spinal cord. However, whether the cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are also activated in response to surgical incision remains to be determined. The present study aimed to investigate the expression pattern of TNF-α and IL-6 in the spinal cord following a deep thoracic incision. After surgical incision, the mRNA levels of TNF-α and IL-6 in the thoracic spinal cord were transiently upregulated as determined by real-time polymerase chain reaction (PCR) assay. However, the activation of IL-6 was detected at 1 h postoperatively, which was earlier compared to that of TNF-α, observed at 6 h postoperatively. The activated TNF-α was mainly localized in the neurons, but not in microglia or astrocytes as determined by immunohistochemistry and confocal microscopy. However, the increased IL-6-immunoreactivity was mainly expressed in blood vessels. The differential upregulation of TNF-α and IL-6 induced by incision suggests that the proinflammatory cytokines may play different roles in the development of surgical pain.

Stem/Progenitor Cell Proteoglycans Decorated with 7-D-4, 4-C-3 and 3-B-3(-) Chondroitin Sulphate Motifs Are Morphogenetic Markers Of Tissue Development.

PubMed

Hayes, Anthony J; Smith, Susan M; Caterson, Bruce; Melrose, James

2018-06-11

This study reviewed the occurrence of chondroitin sulphate (CS) motifs 4-C-3, 7-D-4 and 3-B-3(-) which are expressed by progenitor cells in tissues undergoing morphogenesis. These motifs have a transient early expression pattern during tissue development and also appear in mature tissues during pathological remodeling and attempted repair processes by activated adult stem cells. The CS motifs are information and recognition modules, which may regulate cellular behavior and delineate stem cell niches in developmental tissues. One of the difficulties in determining the precise role of stem cells in tissue development and repair processes is their short engraftment period and the lack of specific markers, which differentiate the activated stem cell lineages from the resident cells. The CS sulphation motifs 7-D-4, 4-C-3 and 3-B-3 (-) decorate cell surface proteoglycans on activated stem/progenitor cells and appear to identify these cells in transitional areas of tissue development and in tissue repair and may be applicable to determining a more precise role for stem cells in tissue morphogenesis. This article is protected by copyright. All rights reserved. © 2018 AlphaMed Press.

The Renilla luciferase gene as a reference gene for normalization of gene expression in transiently transfected cells.

PubMed

Jiwaji, Meesbah; Daly, Rónán; Pansare, Kshama; McLean, Pauline; Yang, Jingli; Kolch, Walter; Pitt, Andrew R

2010-12-31

The importance of appropriate normalization controls in quantitative real-time polymerase chain reaction (qPCR) experiments has become more apparent as the number of biological studies using this methodology has increased. In developing a system to study gene expression from transiently transfected plasmids, it became clear that normalization using chromosomally encoded genes is not ideal, at it does not take into account the transfection efficiency and the significantly lower expression levels of the plasmids. We have developed and validated a normalization method for qPCR using a co-transfected plasmid. The best chromosomal gene for normalization in the presence of the transcriptional activators used in this study, cadmium, dexamethasone, forskolin and phorbol-12-myristate 13-acetate was first identified. qPCR data was analyzed using geNorm, Normfinder and BestKeeper. Each software application was found to rank the normalization controls differently with no clear correlation. Including a co-transfected plasmid encoding the Renilla luciferase gene (Rluc) in this analysis showed that its calculated stability was not as good as the optimised chromosomal genes, most likely as a result of the lower expression levels and transfection variability. Finally, we validated these analyses by testing two chromosomal genes (B2M and ActB) and a co-transfected gene (Rluc) under biological conditions. When analyzing co-transfected plasmids, Rluc normalization gave the smallest errors compared to the chromosomal reference genes. Our data demonstrates that transfected Rluc is the most appropriate normalization reference gene for transient transfection qPCR analysis; it significantly reduces the standard deviation within biological experiments as it takes into account the transfection efficiencies and has easily controllable expression levels. This improves reproducibility, data validity and most importantly, enables accurate interpretation of qPCR data.

Characterization of complex systems using the design of experiments approach: transient protein expression in tobacco as a case study.

PubMed

Buyel, Johannes Felix; Fischer, Rainer

2014-01-31

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

Transient Congenital Hypothyroidism Alters Gene Expression of Glucose Transporters and Impairs Glucose Sensing Apparatus in Young and Aged Offspring Rats.

PubMed

Gholami, Hanieh; Jeddi, Sajad; Zadeh-Vakili, Azita; Farrokhfall, Khadije; Rouhollah, Fatemeh; Zarkesh, Maryam; Ghanbari, Mahboubeh; Ghasemi, Asghar

2017-01-01

Transient congenital hypothyroidism (TCH) could disturb carbohydrate metabolism in adulthood. Aging is associated with increased risk of type 2 diabetes. This study aims to address effects of TCH on mRNA expressions of glucose transporters (GLUTs) and glucokinase (GcK) in islets and insulin target tissues of aged offspring rats. The TCH group received water containing 0.025% 6-propyl-2-thiouracil during gestation. Offspring from control and TCH groups (n=6 in each group) were followed until month 19. Gene expressions of GLUTs and GcK were measured at months 3 and 19. Compared to controls, aged TCH rats had higher GLUT4 expression in heart (4.88 fold) and soleus (6.91 fold), while expression was lower in epididymal fat (12%). In TCH rats, GLUT2 and GcK expressions in islets were lower in young (12% and 10%, respectively) and higher in aged (10.85 and 8.42 fold, respectively) rats. In addition, liver GLUT2 and GcK expressions were higher in young (13.11 and 21.15 fold, respectively) and lower in aged rats (44% and 5%, respectively). Thyroid hormone deficiency during fetal period impaired glucose sensing apparatus and changed glucose transporter expression in insulin-sensitive tissues of aged offspring rats. These changes may contribute to impaired carbohydrate metabolism. © 2017 The Author(s). Published by S. Karger AG, Basel.

Transient severe left ventricular dysfunction following percutaneous patent ductus arteriosus closure in an adult with bicuspid aortic valve: A case report

PubMed Central

HWANG, HUI-JEONG; YOON, KYUNG LIM; SOHN, IL SUK

2016-01-01

The present study reported the case of a 60-year-old female with patent ductus arteriosus (PDA) and a bicuspid aortic valve, who presented with transient severe left ventricular (LV) dysfunction following percutaneous closure of PDA, as identified by speckle tracking analysis. Transient LV dysfunction following PDA closure has previously been reported; however, severe LV dysfunction is rare. In the present case, the combination of a large PDA size, large amount of shunting, LV remodeling and bicuspid aortic valve may have induced serious deterioration of LV function following PDA closure. Furthermore, speckle-tracking echocardiography may be useful in the estimation of functional alterations in the myocardium of the LV following PDA closure. The observations detailed in the present study may improve the understanding of the pathophysiology and myocardial patterns of transient left ventricular dysfunction following PDA closure in adult humans. PMID:26998021

Transient severe left ventricular dysfunction following percutaneous patent ductus arteriosus closure in an adult with bicuspid aortic valve: A case report.

PubMed

Hwang, Hui-Jeong; Yoon, Kyung Lim; Sohn, Il Suk

2016-03-01

The present study reported the case of a 60-year-old female with patent ductus arteriosus (PDA) and a bicuspid aortic valve, who presented with transient severe left ventricular (LV) dysfunction following percutaneous closure of PDA, as identified by speckle tracking analysis. Transient LV dysfunction following PDA closure has previously been reported; however, severe LV dysfunction is rare. In the present case, the combination of a large PDA size, large amount of shunting, LV remodeling and bicuspid aortic valve may have induced serious deterioration of LV function following PDA closure. Furthermore, speckle-tracking echocardiography may be useful in the estimation of functional alterations in the myocardium of the LV following PDA closure. The observations detailed in the present study may improve the understanding of the pathophysiology and myocardial patterns of transient left ventricular dysfunction following PDA closure in adult humans.

Emergent Archetype Hydrological-Biogeochemical Response Patterns in Heterogeneous Catchments

NASA Astrophysics Data System (ADS)

Jawitz, J. W.; Gall, H. E.; Rao, P.

2013-12-01

What can spatiotemporally integrated patterns observed in stream hydrologic and biogeochemical signals generated in response to transient hydro-climatic and anthropogenic forcing tell us about the interactions between spatially heterogeneous soil-mediated hydrological and biogeochemical processes? We seek to understand how the spatial structure of solute sources coupled with hydrologic responses affect observed concentration-discharge (C-Q) patterns. These patterns are expressions of the spatiotemporal structure of solute loads exported from managed catchments, and their likely ecological consequences manifested in receiving water bodies (e.g., wetlands, rivers, lakes, and coastal waters). We investigated the following broad questions: (1) How does the correlation between flow-generating areas and biogeochemical source areas across a catchment evolve under stochastic hydro-climatic forcing? (2) What are the feasible hydrologic and biogeochemical responses that lead to the emergence of the observed archetype C-Q patterns? and; (3) What implications do these coupled dynamics have for catchment monitoring and implementation of management practices? We categorize the observed temporal signals into three archetypical C-Q patterns: dilution; accretion, and constant concentration. We introduce a parsimonious stochastic model of heterogeneous catchments, which act as hydrologic and biogeochemical filters, to examine the relationship between spatial heterogeneity and temporal history of solute export signals. The core concept of the modeling framework is considering the types and degree of spatial correlation between solute source zones and flow generating zones, and activation of different portions of the catchments during rainfall events. Our overarching hypothesis is that each of the archetype C-Q patterns can be generated by explicitly linking landscape-scale hydrologic responses and spatial distributions of solute source properties within a catchment. The model simulations reproduce the three major C-Q patterns observed in published data, offering valuable insight into coupled catchment processes. The findings have important implications for effective catchment management for water quality improvement, and stream monitoring strategies.

Effects from fine muscle and cutaneous afferents on spinal locomotion in cats

PubMed Central

Kniffki, K.-D.; Schomburg, E. D.; Steffens, H.

1981-01-01

1. The effects of chemically activated fine muscle afferents (groups III and IV) and electrically activated cutaneous afferents on motoneuronal discharges were studied before and during fictive locomotion induced pharmacologically by i.v. administration of nialamide and l-DOPA in high spinal cats. Efferent activity was recorded simultaneously from nerve filaments to ipsi- and contralateral extensor and flexor muscles. In addition, intracellular recordings were made from lumbar α-motoneurones. 2. After nialamide but before treatment with l-DOPA, in some cases, transient locomotor-like discharges were induced by an increased activity in fine muscle afferents. The response pattern in nerves to both hind limbs could be different showing e.g. only transient alternating activity between knee flexor and extensor of one limb but not of the other one. 3. Treatment with l-DOPA did not always cause fictive locomotion. Often not all motoneurone pools showed rhythmic activity. In these cases stimulation of group III and IV muscle afferents usually caused transient periodic activity. In cases with apparent rhythmic activity, algesic stimulation of the gastrocnemius—soleus muscle caused an accentuation of the rhythm by a more abrupt transition from the active phase to the non-active interval. Again, the response patterns on both sides were not uniform in all cases. 4. A second type of response to activation of fine muscle afferents had a quite different character: the rhythmic activity was more or less completely overridden by a strong transient tonic hyperactivity or the rhythm was transiently blocked. These phenomena did not occur in the same way in all nerves. 5. Electrical stimulation of cutaneous nerves of the hind limb generally induced the same response pattern as chemical stimulation of the group III and IV muscle afferents. The effects varied depending on the stimulus strength and the nerve. 6. The results revealed that cutaneous and fine muscle afferents not only have similar functions in the reflex control of a limb but also in evocation and modulation of locomotion. Therefore, it is assumed that both types of afferents may serve together as a peripheral feed-back to the spinal locomotor centre. PMID:7320927

Simplified Modeling of Steady-State and Transient Brillouin Gain in Magnetoactive Non-Centrosymmetric Semiconductors

NASA Astrophysics Data System (ADS)

Singh, M.; Aghamkar, P.; Sen, P. K.

With the aid of a hydrodynamic model of semiconductor-plasmas, a detailed analytical investigation is made to study both the steady-state and the transient Brillouin gain in magnetized non-centrosymmetric III-V semiconductors arising from the nonlinear interaction of an intense pump beam with the internally-generated acoustic wave, due to piezoelectric and electrostrictive properties of the crystal. Using the fact that the origin of coherent Brillouin scattering (CBS) lies in the third-order (Brillouin) susceptibility of the medium, we obtained an expression of the gain coefficient of backward Stokes mode in steady-state and transient regimes and studied the dependence of piezoelectricity, magnetic field and pump pulse duration on its growth rate. The threshold-pump intensity and optimum pulse duration for the onset of transient CBS are estimated. The piezoelectricity and externally-applied magnetic field substantially enhances the transient CBS gain coefficient in III-V semiconductors which can be of great use in the compression of scattered pulses.

The influence of computational assumptions on analysing abdominal aortic aneurysm haemodynamics.

PubMed

Ene, Florentina; Delassus, Patrick; Morris, Liam

2014-08-01

The variation in computational assumptions for analysing abdominal aortic aneurysm haemodynamics can influence the desired output results and computational cost. Such assumptions for abdominal aortic aneurysm modelling include static/transient pressures, steady/transient flows and rigid/compliant walls. Six computational methods and these various assumptions were simulated and compared within a realistic abdominal aortic aneurysm model with and without intraluminal thrombus. A full transient fluid-structure interaction was required to analyse the flow patterns within the compliant abdominal aortic aneurysms models. Rigid wall computational fluid dynamics overestimates the velocity magnitude by as much as 40%-65% and the wall shear stress by 30%-50%. These differences were attributed to the deforming walls which reduced the outlet volumetric flow rate for the transient fluid-structure interaction during the majority of the systolic phase. Static finite element analysis accurately approximates the deformations and von Mises stresses when compared with transient fluid-structure interaction. Simplifying the modelling complexity reduces the computational cost significantly. In conclusion, the deformation and von Mises stress can be approximately found by static finite element analysis, while for compliant models a full transient fluid-structure interaction analysis is required for acquiring the fluid flow phenomenon. © IMechE 2014.

Transient enhancement of pericardium, peritoneum, soft tissues and perhaps lymphatics after large doses of contrast administration: an overlooked phenomenon?

PubMed

Behr, Gerald G; Berdon, Walter E; Griscom, N Thorne

2012-06-01

An infant with complex cyanotic congenital heart disease was recently encountered whose radiographs seemed to show enhancement of pericardium, peritoneal mesothelium and body wall fascial planes without enhancement of the liver or spleen after very large doses of intravenous contrast. Although patterns of postcontrast enhancement have been described previously, this pattern seems to be unique. We report the unusual postcontrast opacification pattern and speculate about its underlying mechanism.

The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo.

PubMed

Lewis, Jo E; Brameld, John M; Hill, Phil; Barrett, Perry; Ebling, Francis J P; Jethwa, Preeti H

2015-12-30

The viral 2A sequence has become an attractive alternative to the traditional internal ribosomal entry site (IRES) for simultaneous over-expression of two genes and in combination with recombinant adeno-associated viruses (rAAV) has been used to manipulate gene expression in vitro. To develop a rAAV construct in combination with the viral 2A sequence to allow long-term over-expression of the vgf gene and fluorescent marker gene for tracking of the transfected neurones in vivo. Transient transfection of the AAV plasmid containing the vgf gene, viral 2A sequence and eGFP into SH-SY5Y cells resulted in eGFP fluorescence comparable to a commercially available reporter construct. This increase in fluorescent cells was accompanied by an increase in VGF mRNA expression. Infusion of the rAAV vector containing the vgf gene, viral 2A sequence and eGFP resulted in eGFP fluorescence in the hypothalamus of both mice and Siberian hamsters, 32 weeks post infusion. In situ hybridisation confirmed that the location of VGF mRNA expression in the hypothalamus corresponded to the eGFP pattern of fluorescence. The viral 2A sequence is much smaller than the traditional IRES and therefore allowed over-expression of the vgf gene with fluorescent tracking without compromising viral capacity. The use of the viral 2A sequence in the AAV plasmid allowed the simultaneous expression of both genes in vitro. When used in combination with rAAV it resulted in long-term over-expression of both genes at equivalent locations in the hypothalamus of both Siberian hamsters and mice, without any adverse effects. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Neurogenin 3 Expressing Cells in the Human Exocrine Pancreas Have the Capacity for Endocrine Cell Fate

PubMed Central

Gomez, Danielle L.; O’Driscoll, Marci; Sheets, Timothy P.; Hruban, Ralph H.; Oberholzer, Jose; McGarrigle, James J.; Shamblott, Michael J.

2015-01-01

Neurogenin 3 (NGN3) is necessary and sufficient for endocrine differentiation during pancreatic development and is expressed by a population of progenitor cells that give rise exclusively to hormone-secreting cells within islets. NGN3 protein can be detected in the adult rodent pancreas only following certain types of injury, when it is transiently expressed by exocrine cells undergoing reprogramming to an endocrine cell fate. Here, NGN3 protein can be detected in 2% of acinar and duct cells in living biopsies of histologically normal adult human pancreata and 10% in cadaveric biopsies of organ donor pancreata. The percentage and total number of NGN3+ cells increase during culture without evidence of proliferation or selective cell death. Isolation of highly purified and viable NGN3+ cell populations can be achieved based on coexpression of the cell surface glycoprotein CD133. Transcriptome and targeted expression analyses of isolated CD133+ / NGN3+ cells indicate that they are distinct from surrounding exocrine tissue with respect to expression phenotype and Notch signaling activity, but retain high level mRNA expression of genes indicative of acinar and duct cell function. NGN3+ cells have an mRNA expression profile that resembles that of mouse early endocrine progenitor cells. During in vitro differentiation, NGN3+ cells express genes in a pattern characteristic of endocrine development and result in cells that resemble beta cells on the basis of coexpression of insulin C-peptide, chromogranin A and pancreatic and duodenal homeobox 1. NGN3 expression in the adult human exocrine pancreas marks a dedifferentiating cell population with the capacity to take on an endocrine cell fate. These cells represent a potential source for the treatment of diabetes either through ex vivo manipulation, or in vivo by targeting mechanisms controlling their population size and endocrine cell fate commitment. PMID:26288179

Neuron-Enriched Gene Expression Patterns are Regionally Anti-Correlated with Oligodendrocyte-Enriched Patterns in the Adult Mouse and Human Brain

PubMed Central

Tan, Powell Patrick Cheng; French, Leon; Pavlidis, Paul

2013-01-01

An important goal in neuroscience is to understand gene expression patterns in the brain. The recent availability of comprehensive and detailed expression atlases for mouse and human creates opportunities to discover global patterns and perform cross-species comparisons. Recently we reported that the major source of variation in gene transcript expression in the adult normal mouse brain can be parsimoniously explained as reflecting regional variation in glia to neuron ratios, and is correlated with degree of connectivity and location in the brain along the anterior-posterior axis. Here we extend this investigation to two gene expression assays of adult normal human brains that consisted of over 300 brain region samples, and perform comparative analyses of brain-wide expression patterns to the mouse. We performed principal components analysis (PCA) on the regional gene expression of the adult human brain to identify the expression pattern that has the largest variance. As in the mouse, we observed that the first principal component is composed of two anti-correlated patterns enriched in oligodendrocyte and neuron markers respectively. However, we also observed interesting discordant patterns between the two species. For example, a few mouse neuron markers show expression patterns that are more correlated with the human oligodendrocyte-enriched pattern and vice-versa. In conclusion, our work provides insights into human brain function and evolution by probing global relationships between regional cell type marker expression patterns in the human and mouse brain. PMID:23440889

Neuron-Enriched Gene Expression Patterns are Regionally Anti-Correlated with Oligodendrocyte-Enriched Patterns in the Adult Mouse and Human Brain.

PubMed

Tan, Powell Patrick Cheng; French, Leon; Pavlidis, Paul

2013-01-01

An important goal in neuroscience is to understand gene expression patterns in the brain. The recent availability of comprehensive and detailed expression atlases for mouse and human creates opportunities to discover global patterns and perform cross-species comparisons. Recently we reported that the major source of variation in gene transcript expression in the adult normal mouse brain can be parsimoniously explained as reflecting regional variation in glia to neuron ratios, and is correlated with degree of connectivity and location in the brain along the anterior-posterior axis. Here we extend this investigation to two gene expression assays of adult normal human brains that consisted of over 300 brain region samples, and perform comparative analyses of brain-wide expression patterns to the mouse. We performed principal components analysis (PCA) on the regional gene expression of the adult human brain to identify the expression pattern that has the largest variance. As in the mouse, we observed that the first principal component is composed of two anti-correlated patterns enriched in oligodendrocyte and neuron markers respectively. However, we also observed interesting discordant patterns between the two species. For example, a few mouse neuron markers show expression patterns that are more correlated with the human oligodendrocyte-enriched pattern and vice-versa. In conclusion, our work provides insights into human brain function and evolution by probing global relationships between regional cell type marker expression patterns in the human and mouse brain.