scholarly journals Molecular cloning of the mouse homologue of Rab3c

2001 ◽  
Vol 27 (1) ◽  
pp. 117-122 ◽  
Author(s):  
NJ Pavlos ◽  
J Xu ◽  
JM Papadimitriou ◽  
MH Zheng
Keyword(s):  
Skeletal Development ◽  
In Situ Hybridisation ◽  
Matrix Vesicles ◽  
Matrix Vesicle ◽  
Predicted Amino Acid Sequence ◽  
Multiple Sequence ◽  
Intracellular Vesicle ◽  
Intracellular Vesicle Transport ◽  
The Brain

Small GTP-binding proteins of the Rab subfamily are key regulators of intracellular vesicle transport. Here we report the isolation of a cDNA clone encoding the complete Rab3c isoform from mouse embryo using a degenerative PCR-based approach. Multiple sequence alignment revealed that the predicted amino acid sequence was identical to the previously identified rat Rab3c isoform and 98% identical to the published bovine Rab3c GTPase from brain. Furthermore by in situ hybridisation, Rab3c mRNA was detectable within various regions of the brain, cartilage and highly enriched within intestinal villi of foetal tissues. Chondrocytes in the hypertrophic zone, but not reserve or proliferative zones, expressed high levels of Rab3c. This pattern of expression corresponds with the genesis of matrix vesicles during endochondral ossification.In all, our results suggest that in addition to its functional role during regulated secretion in brain, Rab3c may play a part in matrix vesicle trafficking during skeletal development.

Expression of p53 during mouse embryogenesis

Development ◽  
1991 ◽  
Vol 113 (3) ◽  
pp. 857-865 ◽  
Author(s):  
P. Schmid ◽  
A. Lorenz ◽  
H. Hameister ◽  
M. Montenarh
Keyword(s):  
Gene Expression ◽  
Cellular Differentiation ◽  
Cellular Proliferation ◽  
In Situ Hybridisation ◽  
Terminal Differentiation ◽  
Mouse Embryogenesis ◽  
Differentiated Cells ◽  
The Brain ◽  
P53 Mrna

By in situ hybridisation we have examined the expression of p53 during mouse embryogenesis from day 8.5 to day 18.5 post coitum (p.c.). High levels of p53 mRNA were detected in all cells of the day 8.5 p.c. and 10.5 p.c. mouse embryo. However, at later stages of development, expression became more pronounced during differentiation of specific tissues e.g. of the brain, liver, lung, thymus, intestine, salivary gland and kidney. In cells undergoing terminal differentiation, the level of p53 mRNA declined strongly. In the brain, hybridisation signals were also observed in postmitotic but not yet terminally differentiated cells. Therefore, gene expression of p53 does not appear to be linked with cellular proliferation in this organ. A proposed role for p53 in cellular differentiation is discussed.

scholarly journals Follistatin (FS) in human cerebrospinal fluid and regulation of FS expression in a mouse model of meningitis

2000 ◽  
pp. 809-816 ◽  
Author(s):  
U Michel ◽  
S Ebert ◽  
O Schneider ◽  
Y Shintani ◽  
S Bunkowski ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Cerebrospinal Fluid ◽  
Specific Binding ◽  
In Situ Hybridisation ◽  
Viral Meningitis ◽  
Csf Analysis ◽  
Mrna And Protein Expression ◽  
Leukocyte Counts ◽  
The Brain

OBJECTIVE: Follistatin (FS) is the specific binding protein of activin and expression of both factors is regulated by inflammatory agents. Therefore, FS concentrations were determined in cerebrospinal fluid (CSF) of patients with bacterial and viral meningitis or multiple sclerosis (MS), as well as in the CSF of patients without meningial inflammation or autoimmune diseases. Furthermore, a mouse pneumococcal meningitis model was used to localise the cellular sources of FS in brains of normal and meningitic mice. METHODS: FS concentrations in CSF were determined by ELISA; FS in mice was localised by in situ hybridisation and immunohistochemistry. RESULTS: FS concentrations were > or =0.4 microg/l in 22 of 66 CSF samples of meningitis patients versus 2 of 27 CSF samples from patients with multiple sclerosis (P<0.05) and 2 of 41 CSF specimen from patients without neuroinflammatory diseases (P<0.01). In the CSF of patients with meningitis, the concentration of FS was correlated with total protein (P<0.005) and lactate concentrations (P<0.05), but not with leukocyte counts, interval between onset of disease and CSF analysis, or clinical outcome. The CSF-to-serum ratios of FS and albumin also correlated significantly (P<0.0005). In some patients with meningitis the CSF-to-serum ratios suggested that the elevated FS in CSF did not originate from serum alone. FS was localised in mice brains to neurones of the hippocampus, dentate gyrus, neocortex, and to the choroid plexus. Analyses of brains and other organs from uninfected and infected animals sacrificed 6-36 h after infection did not reveal any obvious differences in the distribution and intensity of FS mRNA and protein expression. CONCLUSIONS: The concentration of FS in humans is elevated during meningitis. In some patients the increase is caused by a release of FS from brain into CSF. Data from the mouse meningitis model suggest that increased CSF concentrations of FS in meningitis appear not to be accompanied by an elevated number of cells containing FS mRNA or protein in the brain.

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.

scholarly journals Single-transcript multiplex in situ hybridisation reveals unique patterns of dystrophin isoform expression in the developing mammalian embryo

2020 ◽  
Vol 5 ◽  
pp. 76
Author(s):  
John C. W. Hildyard ◽  
Abbe H. Crawford ◽  
Faye Rawson ◽  
Dominique O. Riddell ◽  
Rachel C. M. Harron ◽  
...  
Keyword(s):  
Expression Patterns ◽  
In Situ Hybridisation ◽  
Dystrophin Gene ◽  
Tissue Expression ◽  
Mrna Isoforms ◽  
Mammalian Embryo ◽  
Isoform Expression ◽  
Single Transcript ◽  
The Brain

Background: The dystrophin gene has multiple isoforms: full-length dystrophin (dp427) is principally known for its expression in skeletal and cardiac muscle, but is also expressed in the brain, and several internal promoters give rise to shorter, N-terminally truncated isoforms with wider tissue expression patterns (dp260 in the retina, dp140 in the brain and dp71 in many tissues). These isoforms are believed to play unique cellular roles both during embryogenesis and in adulthood, but their shared sequence identity at both mRNA and protein levels makes study of distinct isoforms challenging by conventional methods. Methods: RNAscope is a novel in-situ hybridisation technique that offers single-transcript resolution and the ability to multiplex, with different target sequences assigned to distinct fluorophores. Using probes designed to different regions of the dystrophin transcript (targeting 5', central and 3' sequences of the long dp427 mRNA), we can simultaneously detect and distinguish multiple dystrophin mRNA isoforms at sub-cellular histological levels. We have used these probes in healthy and dystrophic canine embryos to gain unique insights into isoform expression and distribution in the developing mammal. Results: Dp427 is found in developing muscle as expected, apparently enriched at nascent myotendinous junctions. Endothelial and epithelial surfaces express dp71 only. Within the brain and spinal cord, all three isoforms are expressed in spatially distinct regions: dp71 predominates within proliferating germinal layer cells, dp140 within maturing, migrating cells and dp427 appears within more established cell populations. Dystrophin is also found within developing bones and teeth, something previously unreported, and our data suggests orchestrated involvement of multiple isoforms in formation of these tissues. Conclusions: Overall, shorter isoforms appear associated with proliferation and migration, and longer isoforms with terminal lineage commitment: we discuss the distinct structural contributions and transcriptional demands suggested by these findings.

scholarly journals Single-transcript multiplex in situ hybridisation reveals unique patterns of dystrophin isoform expression in the developing mammalian embryo

2020 ◽  
Vol 5 ◽  
pp. 76
Author(s):  
John C. W. Hildyard ◽  
Abbe H. Crawford ◽  
Faye Rawson ◽  
Dominique O. Riddell ◽  
Rachel C. M. Harron ◽  
...  
Keyword(s):  
Expression Patterns ◽  
In Situ Hybridisation ◽  
Dystrophin Gene ◽  
Tissue Expression ◽  
Mrna Isoforms ◽  
Mammalian Embryo ◽  
Isoform Expression ◽  
Single Transcript ◽  
The Brain

Background: The dystrophin gene has multiple isoforms: full-length dystrophin (dp427) is principally known for its expression in skeletal and cardiac muscle, but is also expressed in the brain, and several internal promoters give rise to shorter, N-terminally truncated isoforms with wider tissue expression patterns (dp260 in the retina, dp140 in the brain and dp71 in many tissues). These isoforms are believed to play unique cellular roles both during embryogenesis and in adulthood, but their shared sequence identity at both mRNA and protein levels makes study of distinct isoforms challenging by conventional methods. Methods: RNAscope is a novel in-situ hybridisation technique that offers single-transcript resolution and the ability to multiplex, with different target sequences assigned to distinct fluorophores. Using probes designed to different regions of the dystrophin transcript (targeting 5', central and 3' sequences of the long dp427 mRNA), we can simultaneously detect and distinguish multiple dystrophin mRNA isoforms at sub-cellular histological levels. We have used these probes in healthy and dystrophic canine embryos to gain unique insights into isoform expression and distribution in the developing mammal. Results: Dp427 is found in developing muscle as expected, apparently enriched at nascent myotendinous junctions. Endothelial and epithelial surfaces express dp71 only. Within the brain and spinal cord, all three isoforms are expressed in spatially distinct regions: dp71 predominates within proliferating germinal layer cells, dp140 within maturing, migrating cells and dp427 appears within more established cell populations. Dystrophin is also found within developing bones and teeth, something previously unreported, and our data suggests orchestrated involvement of multiple isoforms in formation of these tissues. Conclusions: Overall, shorter isoforms appear associated with proliferation and migration, and longer isoforms with terminal lineage commitment: we discuss the distinct structural contributions and transcriptional demands suggested by these findings.

scholarly journals Identification of cells expressing Calcitonins A and B, PDF and ACP in Locusta migratoria using cross-reacting antisera and in situ hybridization

2021 ◽  
Author(s):  
Jan Adrianus Veenstra
Keyword(s):  
In Situ Hybridization ◽  
Locusta Migratoria ◽  
In Situ Hybridisation ◽  
Disulfide Bridge ◽  
Neuroendocrine Cells ◽  
Positive Staining ◽  
Diuretic Hormone ◽  
Pigment Dispersing Factor ◽  
The Brain

This work was initiated because an old publication suggested that electrocoagulation of four paraldehyde fuchsin positive cells in the brain of Locusta migratoria might produce a diuretic hormone, the identity of which remains unknown, since none of the antisera to the various putative Locusta diuretic hormones recognizes these cells. The paraldehyde fuchsin positive staining suggests a peptide with a disulfide bridge and the recently identified Locusta calcitonins have both a disulfide bridge and are structurally similar to calcitonin-like diuretic hormone. In situ hybridization and antisera raised to calcitonin-A and -B were used to show were these peptides are expressed in Locusta. Calcitonin-A is produced by neurons and neuroendocrine cells that were previously shown to be immunoreactive to an antiserum to pigment dispersing factor (PDF). The apparent PDF-immunoreactivity in these neurons and neuroendocrine cells is due to crossreactivity with the calcitonin-A precursor. As confirmed by both an PDF-precursor specific antiserum and in situ hybridisation, those calcitonin-A expressing cells do not express PDF. Calcitonin B is expressed by numerous enteroendocrine cells in the midgut as well as the midgut caeca. A guinea pig antiserum to calcitonin A seemed quite specific as it recognized only the calcitonin A expressing cells. However, rabbit antisera to calcitonin-A and-B both crossreacted with neuroendocrine cells in the brain that produce ACP, this is almost certainly due to the common C-terminal dipeptide SPamide that is shared between Locusta calcitonin-A, calcitonin-B and ACP.

Freeze-etch studies of epiphyseal growth plate cartilage reveal matrix vesicles associated with calcification

1978 ◽  
Vol 36 (2) ◽  
pp. 670-671
Author(s):  
Russell N. A. Cecil ◽  
H. Clarke Anderson
Keyword(s):  
Chromic Acid ◽  
Light And Electron Microscopy ◽  
Matrix Vesicles ◽  
Matrix Vesicle ◽  
Glycerol Solution ◽  
Sprague Dawley ◽  
Epiphyseal Growth Plate ◽  
Growth Plates ◽  
Sprague Dawley Rats ◽  
Tibial Epiphyseal

Unfixed proximal tibial epiphyseal growth plates were studied by freeze-etch to confirm the presence of extracellular calcifying matrix vesicles and to determine the substructure of matrix vesicle membranes as compared to plasma and other membranes of intact chondrocytes. Growth plates from 6-10 week old Sprague-Dawley rats were cut into 1x3 mm blocks whose long dimension was oriented either perpendicular or parallel to the long axis of the tibia. Some blocks were fixed at pH 7. 0 in 0. 2M cacodylate - buffered 2. 5% glutaraldehyde for 1 hour at 4ÅC. The blocks were immersed in 30% glycerol solution at 4ÅC for 1 hour, frozen in liquid nitrogen, and then fractured, etched for 2 minutes, and coated with platinum, carbon and 0. 2% Formvar solution. The replicas were cleaned with chromic acid, floated onto Formvar coated grids, and examined with a Phillips EM 300 electron microscope.Fixed and unfixed specimens appeared similar in ultrastructure. Chondrocytes, matrix, and matrix vesicles were identified. In specimens fractured parallel to the long axis of the tibia, the reserve, proliferative, hypertrophic, and calcifying zones could be discerned as described by light and electron microscopy.

Bio-P and non-bio-P bacteria identification by a novel microbial approach

1999 ◽  
Vol 39 (6) ◽  
pp. 13-20 ◽  
Author(s):  
Philip L. Bond ◽  
Jürg Keller ◽  
Linda L. Blackall
Keyword(s):  
In Situ Hybridisation ◽  
Microbial Populations ◽  
Biological Phosphorus Removal ◽  
Gram Positive Bacteria ◽  
P Content ◽  
Identification Of Bacteria ◽  
Widespread Belief ◽  
Biological Phosphorus ◽  
P Removal

Culturing bacteria from activated sludge with enhanced biological phosphorus removal (EBPR) has strongly implicated Acinetobacter with the process. However, using fluorescent in-situ hybridisation (FISH) probing to analyse microbial populations, we have shown evidence opposing this widespread belief. We describe the phosphorus (P) removing performance and microbial population analyses of sludges obtained in a laboratory scale EBPR reactor. Two sludges with extremely high P removing capabilities were examined, the P content of these sludges was 8.6% (P sludge) and 12.3% (S sludge) of the MLSS. Identification of bacteria using FISH probing indicated both sludges were dominated by microbes from the beta proteobacteria and high mol% G+C Gram positive bacteria. Acinetobacter could make up only a small proportion of the cells in these sludges. Sludge with extremely poor P removal (P content of 1.5%, referred to as T sludge) was then generated by reducing the P in the influent. Bacteria resembling the G-bacteria became abundant in this sludge and these were identified using FISH probing. The anaerobic transformations of the T and P sludges correlated well with that of the non-EBPR and EBPR biological models respectively, indicating that bacteria in the T sludge have the potential to inhibit P removal in EBPR systems.

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).

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