Home About us Contact
|
Home About us Contact | ||
|
|
||
Developmental Regulation (developmental + regulation)
Selected AbstractsModeling the Inhibitor Activity and Relative Binding Affinities in Enzyme-Inhibitor-Protein Systems: Application to Developmental Regulation in a PG-PGIP SystemBIOTECHNOLOGY PROGRESS, Issue 3 2004Wayne W. Fish Within a number of classes of hydrolytic enzymes are certain enzymes whose activity is modulated by a specific inhibitor-protein that binds to the enzyme and forms an inactive complex. One unit of a specific inhibitor-protein activity is often defined as the amount necessary to inhibit one unit of its target enzyme by 50 %. No objective quantitative means is available to determine this point of 50 % inhibition in crude systems such as those encountered during purification. Two models were derived: the first model is based on an irreversible binding approximation, and the second, or equilibrium, model is based on reversible binding. The two models were validated using the inhibition data for the polygalacturonase-polygalacturonase-inhibiting protein (PG-PGIP) system. Theory and experimental results indicate that the first model can be used for inhibitor protein activity determination and the second model can be used for inhibitor protein activity determination as well as for comparison of association constants among enzymes and their inhibitor-proteins from multiple sources. The models were used to identify and further clarify the nature of a differential regulation of expression of polygalacturonase-inhibiting protein in developing cantaloupe fruit. These are the first relations that provide for an objective and quantitative determination of inhibitor-protein activity in both pure and crude systems. Application of these models should prove valuable in gaining insights into regulatory mechanisms and enzyme-inhibitor-protein interactions. [source] Zebrafish E-cadherin: Expression during early embryogenesis and regulation during brain developmentDEVELOPMENTAL DYNAMICS, Issue 2 2001Sherry G. Babb Abstract Zebrafish E-cadherin (cdh1) cell adhesion molecule cDNAs were cloned. We investigated spatial and temporal expression of cdh1 during early embryogenesis. Expression was observed in blastomeres, the anterior mesoderm during gastrulation, and developing epithelial structures. In the developing nervous system, cdh1 was detected at the pharyngula stage (24 hpf) in the midbrain-hindbrain boundary (MHB). Developmental regulation of MHB formation involves wnt1 and pax2.1. wnt1 expression preceded cdh1 expression during MHB formation, and cdh1 expression in the MHB was dependent on normal development of this structure. © 2001 Wiley-Liss, Inc. [source] Developmental regulation of the glyoxylate cycle in the human pathogen Penicillium marneffeiMOLECULAR MICROBIOLOGY, Issue 6 2006David Cánovas Summary Penicillium marneffei is a thermally dimorphic opportunistic human pathogen with a saprophytic filamentous hyphal form at 25°C and a pathogenic unicellular yeast form at 37°C. During infection. P. marneffei yeast cells exist intracellularly in macrophages. To cope with nutrient deprivation during the infection process, a number of pathogens employ the glyoxylate cycle to utilize fatty acids as carbon sources. The genes which constitute this pathway have been implicated in pathogenesis. To investigate acetate and fatty acid utilization, the acuD gene encoding a key glyoxylate cycle enzyme (isocitrate lyase) was cloned. The acuD gene is regulated by both carbon source and temperature in P. marneffei, being strongly induced at 37°C even in the presence of a repressing carbon source such as glucose. When introduced into the non-pathogenic monomorphic fungus Aspergillus nidulans, the P. marneffei acuD promoter only responds to carbon source. Similarly, when the A. nidulans acuD promoter is introduced into P. marneffei it only responds to carbon source suggesting that P. marneffei possesses both cis elements and trans -acting factors to control acuD by temperature. The Zn(II)2Cys6 DNA binding motif transcriptional activator FacB was cloned and is responsible for carbon source-, but not temperature-, dependent induction of acuD. The expression of acuD at 37°C is induced by AbaA, a key regulator of morphogenesis in P. marneffei, but deletion of abaA does not completely eliminate temperature-dependent induction, suggesting that acuD and the glyoxylate cycle are regulated by a complex network of factors in P. marneffei which may contribute to its pathogenicity. [source] Developmental regulation of neuron-specific P2X3 receptor expression in the rat cochleaTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 2 2005Lin-Chien Huang Abstract ATP-gated ion channels assembled from P2X3 receptor (P2X3R) subunits contribute to neurotransmission and neurotrophic signaling, associated with neurite development and synaptogenesis, particularly in peripheral sensory neurons. Here, P2X3R expression was characterized in the rat cochlea from embryonic day 16 (E16) to adult (P49,56), using RT-PCR and immunohistochemistry. P2X3R mRNA was strongly expressed in the cochlea prior to birth, declined to a minimal level at P14, and was absent in adult tissue. P2X3R protein expression was confined to spiral ganglion neurons (SGN) within Rosenthal's canal of the cochlea. At E16, immunolabeling was detected in the SGN neurites, but not the distal neurite projection within the developing sensory epithelium (greater epithelial ridge). From E18, the immunolabeling was observed in the peripheral neurites innervating the inner hair cells but was reduced by P6. However, from P2,8, immunolabeling of the SGN neurites extended to include the outer spiral bundle fiber tract beneath the outer hair cells. This labeling of type II SGN afferent fiber declined after P8. By P14, all synaptic terminal immunolabeling in the organ of Corti was absent, and SGN cell body labeling was minimal. In adult cochlear tissue, P2X3R immunolabeling was not detected. Noise exposure did not induce P2X3R expression in the adult cochlea. These data indicate that ATP-gated ion channels incorporating P2X3R subunit expression are specifically targeted to the afferent terminals just prior to the onset of hearing, and likely contribute to the neurotrophic signaling which establishes functional auditory neurotransmission. J. Comp. Neurol. 484:133,143, 2005. © 2005 Wiley-Liss, Inc. [source] Sdmg1 is a component of secretory granules in mouse secretory exocrine tissuesDEVELOPMENTAL DYNAMICS, Issue 1 2009Diana Best Abstract Sdmg1 is a conserved eukaryotic transmembrane protein that is mainly expressed in the gonads where it may have a role in mediating signaling between somatic cells and germ cells. In this study we demonstrate that secretory exocrine cells in the pancreas, salivary gland, and mammary gland also express Sdmg1. Furthermore, we show that Sdmg1 expression is up-regulated during pancreas development when regulated secretory granules start to appear, and that Sdmg1 colocalizes with secretory granule markers in adult pancreatic acinar cells. In addition, we show that Sdmg1 co-purifies with secretory granules during subcellular fractionation of the pancreas and that Sdmg1 and the secretory granule marker Vamp2 are localized to distinct subdomains in the secretory granule membrane. These data suggest that Sdmg1 is a component of regulated secretory granules in exocrine secretory cells and that the developmental regulation of Sdmg1 expression is related to a role for Sdmg1 in post-Golgi membrane trafficking. Developmental Dynamics 238:223,231, 2009. © 2008 Wiley-Liss, Inc. [source] Spatiotemporal distribution of heparan sulfate epitopes during myogenesis and synaptogenesis: A study in developing mouse intercostal muscleDEVELOPMENTAL DYNAMICS, Issue 1 2002Guido J. Jenniskens Abstract Formation of a basal lamina (BL) ensheathing developing skeletal muscle cells is one of the earliest events in mammalian skeletal muscle myogenesis. BL-resident heparan sulfate proteoglycans have been implicated in various processes during myogenesis, including synaptic differentiation. However, attention has focused on the proteoglycan protein core, ignoring the glycosaminoglycan moiety mainly because of a lack of appropriate tools. Recently, we selected a panel of anti,heparan sulfate antibodies applied here to study the spatiotemporal distribution of specific heparan sulfate (HS) epitopes during myogenesis. In mouse intercostal muscle at embryonic day (E14), formation of acetylcholine receptor clusters at synaptic sites coincides with HS deposition. Although some HS epitopes show a general appearance throughout the BL, one epitope preferably clusters at synaptic sites but does so only from E16 onward. During elongation and maturation of primary myotubes, a process preceding secondary myotube development, significant changes in the HS epitope constitution of both synaptic and extrasynaptic BL were observed. As a whole, the data presented here strengthen previous observations on developmental regulation by BL components, and add to the putative roles of specific HS epitopes in myogenesis and synaptogenesis. © 2002 Wiley-Liss, Inc. [source] Mouse Th17 cells: Current understanding of their generation and regulationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2009Chen Dong Abstract IL-17-expressing CD4+ T cells have been recently recognized as a new subset of Th cells, namely Th17 cells. Considerable progress has been made in understanding the developmental regulation of mouse Th17 cells. Here, I summarize this knowledge and discuss on the relationship of Th17 with regulatory and follicular Th cells. [source] Neuroligin-3 is a neuronal adhesion protein at GABAergic and glutamatergic synapsesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2007Elaine C. Budreck Abstract Synaptic adhesion molecules are thought to play a critical role in the formation, function and plasticity of neuronal networks. Neuroligins (NL1,4) are a family of presumptive postsynaptic cell adhesion molecules. NL1 and NL2 isoforms are concentrated at glutamatergic and GABAergic synapses, respectively, but the cellular expression and synaptic localization of the endogenous NL3 and NL4 isoforms are unknown. We generated a panel of NL isoform-specific antibodies and examined the expression, developmental regulation and synaptic specificity of NL3. We found that NL3 was enriched in brain, where NL3 protein levels increased during postnatal development, coinciding with the peak of synaptogenesis. Subcellular fractionation revealed a concentration of NL3 in synaptic plasma membranes and postsynaptic densities. In cultured hippocampal neurons, endogenous NL3 was highly expressed and was localized at both glutamatergic and GABAergic synapses. Clustering of NL3 in hippocampal neurons by neurexin-expressing cells resulted in coaggregation of NL3 with glutamatergic and GABAergic scaffolding proteins. Finally, individual synapses contained colocalized NL2 and NL3 proteins, and coimmunoprecipitation studies revealed the presence of NL1,NL3 and NL2,NL3 complexes in brain extracts. These findings suggest that rodent NL3 is a synaptic adhesion molecule that is a shared component of glutamatergic and GABAergic synapses. [source] Visualization of corticofugal projections during early cortical development in a ,-GFP-transgenic mouseEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2007Erin C. Jacobs Abstract The first postmitotic neurons in the developing neocortex establish the preplate layer. These early-born neurons have a significant influence on the circuitry of the developing cortex. However, the exact timing and trajectory of their projections, between cortical hemispheres and intra- and extra-cortical regions, remain unresolved. Here, we describe the creation of a transgenic mouse using a 1.3 kb golli promoter element of the myelin basic protein gene to target expression of a ,,green fluorescent protein (GFP) fusion protein in the cell bodies and processes of pioneer cortical neurons. During embryonic and early neonatal development, the timing and patterning of process extension from these neurons was examined. Analysis of ,-GFP fluorescent fibers revealed that progression of early labeled projections was interrupted unexpectedly by transient pauses at the corticostriatal and telencephalic,diencephalic boundaries before invading the thalamus just prior to birth. After birth the pioneering projections differentially invaded the thalamus, excluding some nuclei, e.g. medial and lateral geniculate, until postnatal days 10,14. Early labeled projections were also found to cross to the contralateral hemisphere as well as to the superior colliculus. These results indicate that early corticothalamic projections appear to pause before invading specific subcortical regions during development, that there is developmental regulation of innervation of individual thalamic nuclei, and that these early-generated neurons also establish early projections to commissural and subcortical targets. [source] Neuronal activity and neurotrophic factors regulate GAD-65/67 mRNA and protein expression in organotypic cultures of rat visual cortexEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2003Silke Patz Abstract Environmental factors are known to regulate the molecular differentiation of neocortical interneurons. Their class-defining transmitter synthetic enzymes are the glutamic acid decarboxylases (GAD); yet, fairly little is known about the developmental regulation of transcription and translation of the GAD-65/67 isoforms. We have characterized the role of neuronal activity, neurotrophins and afferent systems for GAD-65/67 expression in visual cortex in organotypic cultures (OTC) compared with in vivo in order to identify cortex-intrinsic regulatory mechanisms. Spontaneously active OTC prepared at postnatal day 0 displayed from 10 days in vitro (DIV) onwards 12,14% GAD-65/GAD-67 neurons similar to in vivo. However, GAD-65 mRNA was higher, whereas GAD-67 protein was lower, than in vivo. During the first week neurotrophins increased whereas the Trk receptor inhibitor K252a and MEK inhibitors decreased both GAD mRNAs and proteins. After 10 DIV GAD expression no longer depended on neurotrophin signalling. Activity-deprived OTC revealed only 6% GAD-67 neurons and mRNA and protein were reduced by 50%. GAD-65 mRNA was less reduced, but protein was reduced by half, suggesting translational regulation. Upon recovery of activity GAD mRNAs, cell numbers, and both proteins quickly returned to normal and these ,adult' levels were resistant to late-onset deprivation. In 20 DIV activity-deprived OTC, only neurotrophin 4 increased GAD-65/67 mRNAs, rescued the percentage of GAD-67 neurons and increased both proteins in a TrkB-dependent manner. Activity deprivation had thus shifted the period of neurotrophin sensitivity to older ages. The results suggested neuronal activity as a major regulator differentially affecting transcription and translation of the GAD isoforms. The early presence of neuronal activity promoted the GAD expression in OTC to a neurotrophin-independent state suggesting that neurotrophins play a context-dependent role. [source] Insulin promotes functional induction of silent synapses in differentiating rat neocortical neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2001Daniela Plitzko Abstract Long-term synaptic plasticity is thought to underlie synaptic reorganization phenomena that occur during neocortical development. Recently, it has been proposed, that the functional induction of AMPA receptors at silent glutamatergic synapses is of major importance in activity-dependent, developmental plasticity. To investigate the mechanisms involved in the developmental regulation of silent synapses, we analysed the functional maturation of the thalamocortical projection in culture. A large proportion of the thalamocortical synapses were functionally silent at an early stage in vitro. During further differentiation, the incidence of silent synapses decreased drastically, indicating a conversion of silent into functional synapses. Chronic blockade of spontaneous network activity by addition of tetrodotoxin to the culture medium strongly impaired this developmental maturation. Moreover, the developmental decline in the proportion of silent synapses was dramatically accelerated by chronic addition of the neurotrophic factor, insulin. This effect of insulin was partly dependent on spontaneous activity. Thus, insulin appears to be involved in the modulation of long-term developmental plasticity at immature glutamatergic synapses. [source] Expression of Hoxa-11 and Hoxa-13 in the pectoral fin of a basal ray-finned fish, Polyodon spathula: implications for the origin of tetrapod limbsEVOLUTION AND DEVELOPMENT, Issue 3 2005Brian D. Metscher Summary Paleontological and anatomical evidence suggests that the autopodium (hand or foot) is a novel feature that distinguishes limbs from fins, while the upper and lower limb (stylopod and zeugopod) are homologous to parts of the sarcopterygian paired fins. In tetrapod limb development Hoxa-11 plays a key role in differentiating the lower limb and Hoxa-13 plays a key role in differentiating the autopodium. It is thus important to determine the ancestral functions of these genes in order to understand the developmental genetic changes that led to the origin of the tetrapod autopodium. In particular it is important to understand which features of gene expression are derived in tetrapods and which are ancestral in bony fishes. To address these questions we cloned and sequenced the Hoxa-11 and Hoxa-13 genes from the North American paddlefish, Polyodon spathula, a basal ray-finned fish that has a pectoral fin morphology resembling that of primitive bony fishes ancestral to the tetrapod lineage. Sequence analysis of these genes shows that they are not orthologous to the duplicated zebrafish and fugu genes. This implies that the paddlefish has not duplicated its HoxA cluster, unlike zebrafish and fugu. The expression of Hoxa-11 and Hoxa-13 in the pectoral fins shows two main phases: an early phase in which Hoxa-11 is expressed proximally and Hoxa-13 is expressed distally, and a later phase in which Hoxa-11 and Hoxa-13 broadly overlap in the distal mesenchyme of the fin bud but are absent in the proximal fin bud. Hence the distal polarity of Hoxa-13 expression seen in tetrapods is likely to be an ancestral feature of paired appendage development. The main difference in HoxA gene expression between fin and limb development is that in tetrapods (with the exception of newts) Hoxa-11 expression is suppressed by Hoxa-13 in the distal limb bud mesenchyme. There is, however, a short period of limb bud development where Hoxa-11 and Hoxa-13 overlap similarly to the late expression seen in zebrafish and paddlefish. We conclude that the early expression pattern in tetrapods is similar to that seen in late fin development and that the local exclusion by Hoxa-13 of Hoxa-11 from the distal limb bud is a derived feature of limb developmental regulation. [source] Early evolution of a homeobox gene: the parahox gene Gsx in the Cnidaria and the BilateriaEVOLUTION AND DEVELOPMENT, Issue 4 2003John R. Finnerty Summary Homeobox transcription factors are commonly involved in developmental regulation in diverse eukaryotes, including plants, animals, and fungi. The origin of novel homeobox genes is thought to have contributed to many evolutionary innovations in animals. We perform a molecular phylogenetic analysis of cnox2, the best studied homeobox gene from the phylum Cnidaria, a very ancient lineage of animals. Among three competing hypotheses, our analysis decisively favors the hypothesis that cnox2 is orthologous to the gsx gene of Bilateria, thereby establishing the existence of this specific homeobox gene in the eumetazoan stem lineage, some 650,900 million years ago. We assayed the expression of gsx in the planula larva and polyp of the sea anemone Nematostella vectensis using in situ hybridization and reverse transcriptase polymerase chain reaction. The gsx ortholog of Nematostella, known as anthox2, is expressed at high levels in the posterior planula and the corresponding "head" region of the polyp. It cannot be detected in the anterior planula or the corresponding "foot" region of the polyp. We have attempted to reconstruct the evolution of gsx spatiotemporal expression in cnidarians and bilaterians using a phylogenetic framework. Because of the surprisingly high degree of variability in gsx expression within the Cnidaria, it is currently not possible to infer unambiguously the ancestral cnidarian condition or the ancestral eumetazoan condition for gsx expression. [source] The fabp4 gene of zebrafish (Danio rerio) , genomic homology with the mammalian FABP4 and divergence from the zebrafish fabp3 in developmental expressionFEBS JOURNAL, Issue 6 2007Rong-Zong Liu Teleost fishes differ from mammals in their fat deposition and distribution. The gene for adipocyte-type fatty acid-binding protein (A-FABP or FABP4) has not been identified thus far in fishes. We have determined the cDNA sequence and defined the structure of a fatty acid-binding protein gene (designated fabp4) from the zebrafish genome. The polypeptide sequence encoded by zebrafish fabp4 showed highest identity to the Had -FABP or H6-FABP from Antarctic fishes and the putative orthologs from other teleost fishes (83,88%). Phylogenetic analysis clustered the zebrafish FABP4 with all Antarctic fish H6-FABPs and putative FABP4s from other fishes in a single clade, and then with the mammalian FABP4s in an extended clade. Zebrafish fabp4 was assigned to linkage group 19 at a distinct locus from fabp3. A number of closely linked syntenic genes surrounding the zebrafish fabp4 locus were found to be conserved with human FABP4. The zebrafish fabp4 transcripts showed sequential distribution in the developing eye, diencephalon and brain vascular system, from the middle somitogenesis stage to 48 h postfertilization, whereas fabp3 mRNA was located widely in the embryonic and/or larval central nervous system, retina, myotomes, pancreas and liver from middle somitogenesis to 5 days postfertilization. Differentiation in developmental regulation of zebrafish fabp4 and fabp3 gene transcription suggests distinct functions for these two paralogous genes in vertebrate development. [source] Let's stick together: The role of the Fras1 and Frem proteins in epidermal adhesionIUBMB LIFE, Issue 7 2007Kieran Short Abstract The Fras1 and Frem extracellular matrix proteins play critical roles in epithelial-mesenchymal interaction during embryonic development. Loss of function in humans results in a recessive embryonic blistering disorder called Fraser syndrome. Inactivation of these proteins, or the proteins with which they interact (e.g., Grip1) has also been shown to underlie members of the 'bleb' family of classic mouse mutants which provide a valuable model of Fraser syndrome. Recent studies supporting direct interactions between the Fras1 and Frem proteins, combined with more rigorous elucidation of their developmental regulation, have shed new light on their activity. We summarize the findings to date, bringing new insight into their role in the regulation of epidermal-basement membrane adhesion and organogenesis during development. iubmb Life, 59: 427-435, 2007 [source] The Wnt antagonist secreted frizzled-related protein-1 controls osteoblast and osteocyte apoptosisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Peter V.N. Bodine Abstract Mechanisms controlling human bone formation remain to be fully elucidated. We have used differential display-polymerase chain reaction analysis to characterize osteogenic pathways in conditionally immortalized human osteoblasts (HOBs) representing distinct stages of differentiation. We identified 82 differentially expressed messages and found that the Wnt antagonist secreted frizzled-related protein (sFRP)-1 was the most highly regulated of these. Transient transfection of HOBs with sFRP-1 suppressed canonical Wnt signaling by 70% confirming its antagonistic function in these cells. Basal sFRP-1 mRNA levels increased 24-fold during HOB differentiation from pre-osteoblasts to pre-osteocytes, and then declined in mature osteocytes. This expression pattern correlated with levels of cellular viability such that the pre-osteocytes, which had the highest levels of sFRP-1 mRNA, also had the highest rate of cell death. Basal sFRP-1 mRNA levels also increased 29-fold when primary human mesenchymal stem cells were differentiated to osteoblasts supporting the developmental regulation of the gene. Expression of sFRP-1 mRNA was induced 38-fold following prostaglandin E2 (PGE2) treatment of pre-osteoblasts and mature osteoblasts that had low basal message levels. In contrast, sFRP-1 expression was down-regulated by as much as 80% following transforming growth factor (TGF)-,1 treatment of pre-osteocytes that had high basal mRNA levels. Consistent with this, treatment of pre-osteoblasts and mature osteoblasts with PGE2 increased apoptosis threefold, while treatment of pre-osteocytes with TGF-,1 decreased cell death by 50%. Likewise, over-expression of sFRP-1 in HOBs accelerated the rate of cell death threefold. These results establish sFRP-1 as an important negative regulator of human osteoblast and osteocyte survival. © 2005 Wiley-Liss, Inc. [source] Nicotinic acetylcholine receptor-subunit mRNAs in the mouse superior cervical ganglion are regulated by development but not by deletion of distinct subunit genesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2008G. Putz Abstract Mice with deletions of nicotinic ACh receptor (nAChR) subunit genes are valuable models for studying nAChR functions. We could previously show in the mouse superior cervical ganglion (SCG) that the absence of distinct subunits affects the functional properties of receptors. Here, we have addressed the question of whether deletions of the subunits ,5, ,7, or ,2 are compensated at the mRNA level, monitored by reverse transcription and quantitative real-time polymerase chain reaction. Relative to our reference gene, ,3, which is expressed in all SCG nAChRs, mRNA levels of ,4 showed little change from birth until adult ages in intact ganglia of wild-type mice. In contrast, ,4 declined sharply after birth and was barely detectable in adult animals. ,5, ,7, and ,2 subunit message levels also declined, though more slowly and less completely than ,4. The subunits ,6 and ,3 were detected by conventional polymerase chain reaction at very low levels, if at all, whereas ,2 was never seen in any of our samples. The developmental profile of nAChR mRNA levels in the three knockout strains did not differ markedly from that of wild-type mice. Likewise, message levels of nAChR subunits were similar in cultures prepared from either wild-type or knockout animals. Our observations indicate a developmental regulation of nAChR subunit mRNAs in the SCG of mice after birth that was not affected by the three knockouts under investigation. © 2007 Wiley-Liss, Inc. [source] Mos10 (Vps60) is required for normal filament maturation in Saccharomyces cerevisiaeMOLECULAR MICROBIOLOGY, Issue 5 2003Julia R. Köhler Summary Early pseudohyphal growth of Saccharomyces cerevisiae is well described, and is known to be subject to a complex web of developmental regulation. In maturing filaments, young cells differ significantly from their pseudohyphal progenitors, in their shape, and in their timing and direction of cell division. The changes that occur during filament maturation result in round and oval cells surrounding and covering the pseudohyphal filament. In a screen for mutants that affect this process, a vacuolar protein sorting gene, MOS10 (VPS60), and a gene encoding an , subunit of the proteasome core, PRE9, were isolated. Characterization of the mos10/mos10 phenotype showed that the process of filament maturation is regulated differently from early filamentous growth, and that the requirement for Mos10 is limited to the maturation stage of pseudohyphal development. The mos10/mos10 phenotype is unlikely to be an unspecific effect of disruption of endocytosis or vacuolar protein sorting, because it is not recapitulated by mutants in other genes required for these processes. Disruption of homologues of MOS10, which act as components of the ESCRT-III complex in targeting proteins for vacuolar degradation, results in abnormal early pseudohyphal growth, not in the filament maturation defect seen in mos10/mos10. Thus, Mos10 may function in targeting of specific cargo proteins for degradation, under conditions particular to maturing filaments. [source] The CFTR gene and regulation of its expressionPEDIATRIC PULMONOLOGY, Issue 1 2005Victoria A. McCarthy Abstract The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows clear temporal and developmental regulation of its expression. However, there are few well-defined regulatory elements that control this pattern of expression, and their mechanism of action is poorly understood. We review the structure and organization of the CFTR gene and what is known about its regulation. The CFTR gene promoter is clearly important for maintaining levels of CFTR gene expression, but apparently it does not contain any tissue-specific elements. Thus tissue-specificity is probably controlled by sequences lying elsewhere in this large gene. We discuss data from our group and others implicating additional regions of CFTR in regulatory functions, and evaluate candidate transcription factors that may be involved. Further, we summarize aspects of the regulation of the developmental expression of CFTR. Definition of CFTR gene regulatory elements could be of considerable therapeutic significance, since only a small increase in CFTR expression in the correct cell type could alleviate the disease phenotype. Pediatr Pulmonol © 2005 Wiley-Liss, Inc. [source] In situ analysis of enzymes involved in sucrose to hexose-phosphate conversion during stolon-to-tuber transition of potatoPHYSIOLOGIA PLANTARUM, Issue 2 2002Niek J.G. Appeldoorn An in situ study of enzymes involved in sucrose to hexose-phosphate conversion during in vitro stolon-to-tuber transition of potato (Solanum tuberosum L. cv. Bintje) was employed to follow developmental changes in spatial patterns. In situ activity of the respective enzymes was visualized by specific activity-staining techniques and they revealed distinct spatially and developmentally regulated patterns. Two of the enzymes studied were also subject to in situ investigations at the transcriptional level. During the stages of stolon formation high hexokinase (EC 2.7.1.1) and acid (cell wall-bound) invertase (EC 3.2.1.26) activities were restricted to the mitotically active (sub)apical region, suggesting a possible importance of these enzymes for cell division. At the onset of tuberization sucrose synthase (EC 2.4.1.13) and fructokinase (EC 2.7.1.4) were strongly induced (visualized at transcriptional and translational level) and the acid invertase activities disappeared from the swelling subapical region as expected. The high degree of similarity in the spatial pattern and the temporal induction of sucrose synthase and fructokinase suggests a tightly co-ordinated coarse (up)regulation, which may be subject to a sugar-modulated mechanism(s) by which genes involved in the metabolic sucrose-starch converting potential are co-ordinately regulated during tuber growth. The overall activity of uridine-5-diphosphoglucose pyrophosphorylase (EC 2.7.7.9) was present in all tissues during stolon and tuber development, implying that its coarse control is not subject to (in)direct developmental regulation. [source] Rapid analysis of Jatropha curcas gene functions by virus-induced gene silencingPLANT BIOTECHNOLOGY JOURNAL, Issue 9 2009Jian Ye Summary Jatropha curcas L. is a small, woody tree of the Euphorbiaceae family. This plant can grow on marginal land in the tropical and subtropical regions and produces seeds containing up to 30% oil. Several Asian countries have selected Jatropha for large scale planting as a biodiesel feedstock. Nevertheless, Jatropha also possesses several undesirable traits that may limit its wide adoption. An improved understanding of plant development and the regulation of fatty acid (FA) and triacylglyceride biosynthesis in Jatropha is particularly facilitative for the development of elite crops. Here, we show that a tobacco rattle virus (TRV) vector can trigger virus-induced gene silencing (VIGS) in Jatropha. Our optimized method produced robust and reliable gene silencing in plants agroinoculated with recombinant TRV harbouring Jatropha gene sequences. We used VIGS to investigate possible functions of 13 Jatropha genes of several functional categories, including FA biosynthesis, developmental regulation and toxin biosynthesis, etc. Based on the effects of VIGS on the FA composition of newly emerged leaves, we determined the function of several genes implicated in FA biosynthesis. Moreover, VIGS was able to discriminate independent functions of related gene family members. Our results show that VIGS can be used for high-throughput screening of Jatropha genes whose functions can be assayed in leaves. [source] Proteomic analysis of shoot-borne root initiation in maize (Zea mays L.)PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2006Michaela Sauer Abstract Postembryonically formed shoot-borne roots make up the major backbone of the adult maize root stock. In this study the abundant soluble proteins of the first node (coleoptilar node) of wild-type and mutant rtcs seedlings, which do not initiate crown roots, were compared at two early stages of crown root formation. In Coomassie Bluestained 2-D gels, representing soluble proteins of coleoptilar nodes 5 and 10,days after germination, 146 and 203 proteins were detected, respectively. Five differentially accumulated proteins (>two-fold change; t -test: 95% significance) were identified in 5-day-old and 14 differentially accumulated proteins in 10-day-old coleoptilar nodes of wild-type versus rtcs. All 19 differentially accumulated proteins were identified via ESI MS/MS mass spectrometry. Five differentially accumulated proteins, including a regulatory G-protein and a putative auxin-binding protein, were further analyzed at the RNA expression level. These experiments confirmed differential gene expression and revealed subtle developmental regulation of these genes during early coleoptilar node development. This study represents the first proteomic analysis of shoot-borne root initiation in cereals and will contribute to a better understanding of the molecular basis of this developmental process unique to cereals. [source] GABAB receptor modulation of excitatory and inhibitory synaptic transmission onto rat CA3 hippocampal interneuronsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2003Saobo Lei Hippocampal stratum radiatum inhibitory interneurons receive glutamatergic excitatory innervation via the recurrent collateral fibers of CA3 pyramidal neurons and GABAergic inhibition from other interneurons. We examined both presynaptic- and postsynaptic-GABAB receptor-mediated responses at both synapse types. Postsynaptic GABAB receptor-mediated responses were absent in recordings from young (P16-18) but present in recordings from older animals (P30) suggesting developmental regulation. In young animals, the GABAB receptor agonist, baclofen, inhibited the amplitude of evoked EPSCs and IPSCs, an effect blocked by prior application of the selective antagonist CGP55845. Baclofen enhanced the paired-pulse ratio and coefficient of variation of evoked EPSCs and IPSCs, consistent with a presynaptic mechanism of regulation. In addition, baclofen reduced the frequency of miniature IPSCs but not mEPSCs. However, baclofen reduced the frequency of KCl-induced mEPSCs; an effect blocked by Cd2+, implicating presynaptic voltage-gated Ca2+ channels as a target for baclofen modulation. In contrast, although Cd2+ prevented the KCl-induced increase in mIPSC frequency, it failed to block baclofen's reduction of mIPSC frequency. Whereas N- and P/Q-types of Ca2+ channels contributed equally to GABAB receptor-mediated inhibition of EPSCs, more P/Q-type Ca2+ channels were involved in GABAB receptor-mediated inhibition of IPSCs. Finally, baclofen blocked the frequency-dependent depression of EPSCs and IPSCs, but was less effective at blocking frequency-dependent facilitation of EPSCs. Our results demonstrate that presynaptic GABAB receptors are expressed on the terminals of both excitatory and inhibitory synapses onto CA3 interneurons and that their activation modulates essential components of the release process underlying transmission at these two synapse types. [source] Differential targeting of GSH1 and GSH2 is achieved by multiple transcription initiation: implications for the compartmentation of glutathione biosynthesis in the BrassicaceaeTHE PLANT JOURNAL, Issue 1 2005Andreas Wachter Summary The genome of Arabidopsis thaliana reveals that in this species the enzymes of glutathione biosynthesis, GSH1 and GSH2, are encoded by single genes. In silico analysis predicts proteins with putative plastidic transit peptides (TP) for both genes, but this has not been experimentally verified. Here we report a detailed analysis of the 5,ends of GSH1 and GSH2 mRNAs and demonstrate the subcellular targeting of the proteins encoded by different transcript types. GSH1 transcript analysis revealed two mRNA populations with short and long 5,-UTRs, respectively, both including the entire TP sequence. The ratio of long/total GSH1 transcripts was subject to developmental regulation. Transient transformation experiments with reporter gene fusions, bearing long or short 5,-UTRs, indicated an exclusive targeting of GSH1 to the plastids. Corroborating these results, endogenous and ectopically expressed GSH1 proteins were always present as a single polypeptide species with the size expected for correctly processed GSH1. Finally, the plastidic GSH1 localization was confirmed by immunocytochemistry. Similar to GSH1, multiple transcript populations were found for GSH2. However, here the prevalent shorter transcripts lacked a complete TP sequence. As expected, the large (but less abundant) transcript encoded a plastidic GSH2 protein, whereas GSH2 synthesized from the shorter transcript was targeted to the cytosol. The implications of the results for the compartmentation and regulation of GSH synthesis are discussed. [source] Molecular characterization of two novel deltamethrin-inducible P450 genes from Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2010Hong-Bo Jiang Abstract Two novel P450 genes, CYP6CE1 and CYP6CE2 (GenBank accession number: EF421245 and EF421246), were cloned and characterized from psocid, Liposcelis bostrychophila. CYP6CE1 and CYP6CE2 contain open reading frames of 1,581 and 1,563 nucleotides that encode 527 and 521 amino acid residues, respectively. The putative proteins of CYP6CE1 and CYP6CE2 show predicted molecular weights of 60.76 and 59.83,kDa with a theoretical pI of 8.58 and 8.78, respectively. CYP6CE1 and CYP6CE2 share 74% identity with each other, and the deduced proteins are typical microsomal P450s sharing signature sequences with other insect CYP6 P450s. Both CYP6CE1 and CYP6CE2 share the closest identities with Hodotermopsis sjoestedti CYP6AM1 at 48% among the published sequences. Phylogenetic analysis showed a closer relationship of CYP6CE1 and CYP6CE2 with CYP6 members of other insects than with those from other families. Quantitative real-time RT-PCR showed that both CYP6CE1 and CYP6CE2 are expressed at all developmental stages tested. Interestingly, CYP6CE2 transcripts decreased from the highest in 1st nymph to the lowest in adults, which seemed to suggest developmental regulation. However, neither CYP6CE1 nor CYP6CE2 were stage specific. The CYP6CE1 and CYP6CE2 transcripts in adults increased significantly after deltamethrin exposure. Recombinant protein expression studies are needed to determine the real functions of these proteins. © 2010 Wiley Periodicals, Inc. [source] Peptidoglycan induces loss of a nuclear peptidoglycan recognition protein during host tissue development in a beneficial animal-bacterial symbiosisCELLULAR MICROBIOLOGY, Issue 7 2009Joshua V. Troll Summary Peptidoglycan recognition proteins (PGRPs) are mediators of innate immunity and recently have been implicated in developmental regulation. To explore the interplay between these two roles, we characterized a PGRP in the host squid Euprymna scolopes (EsPGRP1) during colonization by the mutualistic bacterium Vibrio fischeri. Previous research on the squid-vibrio symbiosis had shown that, upon colonization of deep epithelium-lined crypts of the host light organ, symbiont-derived peptidoglycan monomers induce apoptosis-mediated regression of remote epithelial fields involved in the inoculation process. In this study, immunofluorescence microscopy revealed that EsPGRP1 localizes to the nuclei of epithelial cells, and symbiont colonization induces the loss of EsPGRP1 from apoptotic nuclei. The loss of nuclear EsPGRP1 occurred prior to DNA cleavage and breakdown of the nuclear membrane, but followed chromatin condensation, suggesting that it occurs during late-stage apoptosis. Experiments with purified peptidoglycan monomers and with V. fischeri mutants defective in peptidoglycan-monomer release provided evidence that these molecules trigger nuclear loss of EsPGRP1 and apoptosis. The demonstration of a nuclear PGRP is unprecedented, and the dynamics of EsPGRP1 during apoptosis provide a striking example of a connection between microbial recognition and developmental responses in the establishment of symbiosis. [source] | ||||||||||||||||||||||||||||