scholarly journals Patterns of E74A RNA and protein expression at the onset of metamorphosis in Drosophila

Development ◽  
1991 ◽  
Vol 112 (4) ◽  
pp. 981-995 ◽  
Author(s):  
L. Boyd ◽  
E. O'Toole ◽  
C.S. Thummel
Keyword(s):  
Protein Expression ◽  
Polytene Chromosomes ◽  
Protein Accumulation ◽  
Spatial And Temporal Patterns ◽  
Larval Salivary Gland ◽  
Initial Appearance ◽  
Nascent Transcripts ◽  
Post Transcriptional Regulation ◽  
The Brain

Metamorphosis in Drosophila is triggered by a pulse of the steroid hormone ecdysone at the end of larval development. Ecdysone initiates a genetic hierarchy that can be visualized as a series of puffs in the larval salivary gland polytene chromosomes. The E74 gene is responsible for the early ecdysone-inducible puff at position 74EF and encodes two related DNA-binding proteins which appear to play a regulatory role in the hierarchy. Here we describe the spatial and temporal patterns of E74A RNA and protein expression at the onset of metamorphosis. We use in situ hybridization, antibody stains, and western and northern blot analyses to follow E74A expression from its initial appearance as nascent transcripts on the polytene chromosomes, to spliced mRNA, to post-translationally modified nuclear E74A protein. E74A is expressed in a wide variety of late-third instar tissues, suggesting that it plays a broad pleiotropic role in response to the hormone. In early prepupae, when the overall levels of E74A mRNA are decreasing, relatively high levels of E74A RNA persist in the gut, peripodial membranes of the imaginal discs, and proliferation centers of the brain. The spatial distribution of nuclear E74A protein correlates with the RNA distribution with the single exception that no E74A protein can be detected in the proliferation centers of the brain. There is also a temporal discrepancy between E74A mRNA and protein accumulation. The peak of E74A protein induced by the late larval ecdysone pulse follows the peak of E74A mRNA by approximately 2 h. This delay is not seen in 10 h prepupae, when the next pulse of ecdysone induces the simultaneous expression of E74A mRNA and protein. We discuss possible mechanisms for post-transcriptional regulation of E74A expression and suggest that the unusually long and complex 5′ leader in the E74A mRNA may regulate its translation.

scholarly journals Expression of the Cystathionine β Synthase (CBS) Gene During Mouse Development and Immunolocalization in Adult Brain

2003 ◽  
Vol 51 (3) ◽  
pp. 363-371 ◽  
Author(s):  
Karine Robert ◽  
François Vialard ◽  
Eric Thiery ◽  
Kiyoko Toyama ◽  
Pierre-Marie Sinet ◽  
...  
Keyword(s):  
Plasma Concentrations ◽  
Northern Blotting ◽  
Adult Brain ◽  
Spatial And Temporal Patterns ◽  
Mouse Development ◽  
Cellular Expression ◽  
Brain Expression ◽  
Neuronal Processes ◽  
The Brain

Hyperhomocysteinemia, caused by a lack of cystathionine β synthase (CBS), leads to elevated plasma concentrations of homocysteine. This is a common risk factor for atherosclerosis, stroke, and possibly neurodegenerative diseases. However, the mechanisms that link hyperhomocysteinemia due to CBS deficiency to these diseases are still unknown. Early biochemical studies describe developmental and adult patterns of transsulfuration and CBS expression in a variety of species. However, there is incomplete knowledge about the regional and cellular expression pattern of CBS, notably in the brain. To complete the previous data, we used in situ hybridization and Northern blotting to characterize the spatial and temporal patterns of Cbs gene expression during mouse development. In the early stages of development, the Cbs gene was expressed only in the liver and in the skeletal, cardiac, and nervous systems. The expression declined in the nervous system in the late embryonic stages, whereas it increased in the brain after birth, peaking during cerebellar development. In the adult brain, expression was strongest in the Purkinje cell layer and in the hippocampus. Immunohistochemical analyses showed that the CBS protein was localized in most areas of the brain but predominantly in the cell bodies and neuronal processes of Purkinje cells and Ammon's horn neurons.

scholarly journals Targeting cytochrome P450 46A1 and brain cholesterol 24-hydroxylation to treat neurodegenerative diseases

2021 ◽  
Author(s):  
Irina Pikuleva
Keyword(s):  
Cytochrome P450 ◽  
Motor Behavior ◽  
Cholesterol Biosynthesis ◽  
Cholesterol Content ◽  
Brain Diseases ◽  
Protein Accumulation ◽  
Brain Cholesterol ◽  
Tightly Coupled ◽  
The Brain

The brain cholesterol content is determined by the balance between the pathways of in situ biosynthesis and cholesterol elimination via 24-hydroxylation catalyzed by cytochrome P450 46A1 (CYP46A1). Both pathways are tightly coupled and determine the rate of brain cholesterol turnover. Evidence is accumulating that modulation of CYP46A1 activity by gene therapy or pharmacologic means could be beneficial in the case of neurodegenerative and other brain diseases and affect brain processes other than cholesterol biosynthesis and elimination. This minireview summarizes these other processes, most common of which include abnormal protein accumulation, memory, and cognition, motor behavior, gene transcription, protein phosphorylation as well as autophagy and lysosomal processing. The unifying mechanisms, by which these processes could be affected by CYP46A targeting are also discussed.

scholarly journals Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains

2017 ◽  
Author(s):  
Lu Yang ◽  
Joshua S. Titlow ◽  
Darragh Ennis ◽  
Carlas Smith ◽  
Jessica Mitchell ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Single Molecule ◽  
Single Gene ◽  
In Situ Hybridisation ◽  
Brain Structures ◽  
Fluorescence In Situ Hybridisation ◽  
Stems Cells ◽  
Post Transcriptional Regulation ◽  
The Brain

AbstractRNA in situ hybridization can be a powerful method to investigate post-transcriptional regulation, but analysis of intracellular mRNA distributions in thick, complex tissues like the brain poses significant challenges. Here, we describe the application of single-molecule fluorescent in situ hybridization (smFISH) to quantitate primary transcription and post-transcriptional regulation in whole-mount Drosophila larval and adult brains. Combining immunofluorescence and smFISH probes for different regions of a single gene, i.e., exons, 3’UTR, and introns, we show examples of a gene that is regulated post-transcriptionally and one that is regulated at the level of transcription. We also show that the method can be used to co-visualise a variety of different transcripts and proteins in neuronal stems cells as well as deep brain structures such as mushroom body neuropils. Finally, we introduce the use of smFISH as asensitivealternative to conventional antibody labelling to mark specific neural stem cell populations in the brain.

Expression of Blood Coagulation Factor X Is Not Liver-Restricted.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1939-1939
Author(s):  
Shing Jen Tai ◽  
Parker Hudson ◽  
Christian Furlan Freguia ◽  
Katherine A. High
Keyword(s):  
Blood Coagulation ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Factor X ◽  
Spatial And Temporal Patterns ◽  
Adult Mice ◽  
Immuno Histochemistry ◽  
The Brain

Abstract Factor X (FX) is a vitamin K-dependent clotting factor that plays a critical role in blood coagulation by catalyzing the conversion of prothrombin to thrombin. Although predominantly found in liver, expression of FX is not liver-restricted as shown in previous studies (JBC271:2323–2331, 1996; EMBO J.11:467–472, 1992). Expression of FX was also detected in whole mouse embryo extracts by RT-PCR as early as E7.5, prior to the formation of a liver bud (TH84:1023–1030, 2000). Other studies have suggested additional biological functions for FX that are independent of its role in blood coagulation, including stimulation of mitogenic activity in endothelial cells, enhancement of platelet-derived growth factor (PDGF) release from vascular smooth muscle cells, induction of cytokine production, and up-regulation of the early growth response-1 (egr-1) gene transcription. FX-knockout [FX (−/−)] mice generated by our group as well as by Dewerchin et al. showed partial embryonic lethality beginning as early as E10.5 and fatal perinatal bleeding in the remaining FX (−/−) mice surviving to term. However, as for several other coagulation-related knock-out mice, the exact cause of embryonic lethality observed in some FX (−/−) mice remains to be deciphered. In light of these observations, we sought to determine the spatial and temporal patterns of FX expression in both developing and adult mice. Our preliminary studies, utilizing the combined techniques of Northern blot analysis, immuno-histochemistry, and in situ hybridization revealed the following. Northern blot analysis of mRNA isolated from different tissues of wild-type (+/+) adult mice showed FX transcript in multiple tissues including liver, stomach, spleen, lung, colon, ovaries, placenta, and heart (in decreasing levels of FX expression). Results of immuno-histochemistry on selective adult mouse tissues were similar to the results of Northern blot analysis with the exception of kidney, in which we found FX protein in the cortical, but not in the medullary region. Specifically, we noted expression of FX protein in the bronchi/bronchioles of the lung, and selective cells in the myocardium and in pancreas. However, despite detection of FX transcript in spleen and placenta, we failed to detect FX protein in either of these tissues. In developing embryos, immuno-histochemistry revealed expression of FX protein in liver, small intestines, and thymus for wild-type E14.5 paraffin-embedded sagittal sections, and expression of FX protein in liver and selective cells within the brain for E15.5 sagittal sections. Additionally, we carried out in situ hybridization of paraffin-embedded sagittal sections, using digoxigenin-labeled Factor X antisense riboprobe constructed from a 1 kb fragment of the 5′ end of the murine FX cDNA (identical to the probe used in Northern blot). For E12.5 embryos, FX transcript was found predominantly in the liver. For E14.5 embryos, we detected FX transcript not only in the liver, but also in the kidney (specifically in the primitive glomeruli). For E15.5 embryos, we noted FX transcripts in liver, lung, and selective cells in the brain. Already in progress, additional studies including both FX (+/+) and FX (−/−) embryos at earlier stages of development (for example E9.5 to E13.5) and additional adult tissues should provide a more complete delineation of the spatial and temporal patterns of FX expression in mouse development and adulthood. In conclusion, expression of murine FX is not restricted to liver during embryonic development or during adulthood.

scholarly journals Lipopolysaccharide Induction of Tissue Factor Expression in Rabbits

1999 ◽  
Vol 67 (5) ◽  
pp. 2540-2546 ◽  
Author(s):  
Jonathan Erlich ◽  
Colleen Fearns ◽  
John Mathison ◽  
Richard J. Ulevitch ◽  
Nigel Mackman
Keyword(s):  
In Situ Hybridization ◽  
Protein Expression ◽  
Tissue Factor ◽  
Tissue Injury ◽  
Immunohistochemical Analysis ◽  
Cell Types ◽  
Fibrin Deposition ◽  
Mrna And Protein Expression ◽  
The Brain

ABSTRACT Tissue factor (TF) is the major activator of the coagulation protease cascade and contributes to lethality in sepsis. Despite several studies analyzing TF expression in animal models of endotoxemia, there remains debate about the cell types that are induced to express TF in different tissues. In this study, we performed a detailed analysis of the induction of TF mRNA and protein expression in two rabbit models of endotoxemia to better understand the cell types that may contribute to local fibrin deposition and disseminated intravascular coagulation. Northern blot analysis demonstrated that lipopolysaccharide (LPS) increased TF expression in the brain, lung, and kidney. In situ hybridization showed that TF mRNA expression was increased in cells identified morphologically as epithelial cells in the lung and as astrocytes in the brain. In the kidney, in situ hybridization experiments and immunohistochemical analysis showed that TF mRNA and protein expression was increased in renal glomeruli and induced in tubular epithelium. Dual staining for TF and vWF failed to demonstrate TF expression in endothelial cells in LPS-treated animals. These results demonstrate that TF expression is induced in many different cell types in LPS-treated rabbits, which may contribute to local fibrin deposition and tissue injury during endotoxemia.

Substituted N,N-Dimethyl-3-(phenylthio)- and -4-(phenylthio)benzylamines

1992 ◽  
Vol 57 (1) ◽  
pp. 194-203 ◽  
Author(s):  
Karel Šindelář ◽  
Vojtěch Kmoníček ◽  
Marta Hrubantová ◽  
Zdeněk Polívka
Keyword(s):  
Serotonin Receptors ◽  
Hydrobromic Acid ◽  
Antidepressant Activity ◽  
Benzoic Acids ◽  
Lithium Aluminium Hydride ◽  
Acid Chlorides ◽  
Activity Inhibition ◽  
Lithium Aluminium ◽  
The Brain

(Arylthio)benzoic acids IIa - IIe and VIb - VId were transformed via the acid chlorides to the N,N-dimethylamides which were reduced either with diborane "in situ" or with lithium aluminium hydride to N,N-dimethyl-(arylthio)benzylamines Ia - Ie and Vb - Vd. Leuckart reaction of the aldehydes IX and X with dimethylformamide and formic acid afforded directly the amines Va and Ve. Demethylation of the methoxy compounds Ia and Ve with hydrobromic acid resulted in the phenolic amines If and Vf. The most interesting N,N-dimethyl-4-(phenylthio)benzylamine (Va) hydrochloride showed affinity to cholinergic and 5-HT2 serotonin receptors in the rat brain and some properties considered indicative of antidepressant activity (inhibition of serotonin re-uptake in the brain and potentiation of yohimbine toxicity in mice).

scholarly journals Molecular Characterization of hobo-Mediated Inversions in Drosophila melanogaster

Genetics ◽  
1996 ◽  
Vol 144 (2) ◽  
pp. 647-656
Author(s):  
William B Eggleston ◽  
Nac R Rim ◽  
Johng K Lim
Keyword(s):  
X Chromosome ◽  
Polymerase Chain Reaction Amplification ◽  
Polytene Chromosomes ◽  
Chromosomal Inversions ◽  
Hobo Element ◽  
Reaction Amplification ◽  
Notch Locus ◽  
Polymerase Chain

Abstract The structure of chromosomal inversions mediated by hobo transposable elements in the Uc-1 X chromosome was investigated using cytogenetic and molecular methods. Uc-1 contains a phenotypically silent hobo element inserted in an intron of the Notch locus. Cytological screening identified six independent Notch mutations resulting from chromosomal inversions with one breakpoint at cytological position 3C7, the location of Notch. In situ hybridization to salivary gland polytene chromosomes determined that both ends of each inversion contained hobo and Notch sequences. Southern blot analyses showed that both breakpoints in each inversion had hobo-Notch junction fragments indistinguishable in structure from those present in the Uc-1 X chromosome prior to the rearrangements. Polymerase chain reaction amplification of the 12 hobo-Notch junction fragments in the six inversions, followed by DNA sequence analysis, determined that each was identical to one of the two hobo-Notch junctions present in Uc-1. These results are consistent with a model in which hobo-mediated inversions result from homologous pairing and recombination between a pair of hobo elements in reverse orientation.

scholarly journals CHROMOSOMAL SEQUENCES AND INTERISLAND COLONIZATIONS IN HAWAIIAN DROSOPHILA

Genetics ◽  
1983 ◽  
Vol 103 (3) ◽  
pp. 465-482
Author(s):  
Hampton L Carson
Keyword(s):  
Polytene Chromosomes ◽  
Complex Species ◽  
Hawaiian Drosophila ◽  
On This Island ◽  
Break Points ◽  
Chromosomal Sequences ◽  
Founder Events ◽  
Paracentric Inversions ◽  
Single Set

ABSTRACT Of 103 picture-winged Drosophila species endemic to the high Hawaiian islands, all but three are endemic to single islands or island complexes. They are presumed to have evolved in situ on each island. The banding pattern sequences of the five major polytene chromosomes of these species have been mapped to a single set of Standard sequences. Sequential variation among these chromosomes is due to 213 paracentric inversions. An atlas of their break points is provided. Geographical, morphological and behavioral data may be used to supplement the cytological information in tracing ancestry. Starting at the newer end of the archipelago, the 26 species of the Island of Hawaii (less than 700,000 years old) are inferred to have been derived from 19 founders, 15 from the Maui complex, three from Oahu and one from Kauai. The existence of 40 Maui complex species is explicable as resulting from 12 founders, ten from Oahu and two from Kauai. The 29 Oahu species can be explained by 12 founder events, five from Kauai and seven from Maui complex (summary in Figure 5). Although the ancestry of two Kauai species can be traced to newer islands, the ten remaining ones on this island (age about 5.6 million years) are apparently ancient elements in the fauna, relating ultimately to Palearctic continental sources.

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