scholarly journals Distinct distribution of vimentin and cytokeratin in Xenopus oocytes and early embryos

1992 ◽  
Vol 101 (1) ◽  
pp. 151-160 ◽  
Author(s):  
N.P. Torpey ◽  
J. Heasman ◽  
C.C. Wylie
Keyword(s):  
Spinal Cord ◽  
Amino Acid ◽  
Xenopus Oocytes ◽  
Germ Plasm ◽  
Predicted Amino Acid Sequence ◽  
Larval Stages ◽  
Functional Differences ◽  
Early Embryos ◽  
Strong Staining ◽  
Distinct Distribution

We report the identity of a major component of Triton-insoluble extracts from Xenopus oocytes and early embryos. In a previous paper we showed that an antibody, Z9, cross-reacts with two polypeptides from such extracts (Mr 56,000 and 57,000) as well as Xenopus vimentin. Direct microsequencing of the Mr 57,000 protein shows near identity of three tryptic fragments with regions of the predicted amino acid sequence of XCK1(8), a basic cytokeratin whose mRNA is known to be expressed in Xenopus oocytes. We have raised an antibody, CK7, against a fusion protein generated from this cDNA. The specificity of this antibody has been tested using 1- and 2-dimensional immunoblotting, which show that it is specific for the Mr 56,000 and 57,000 proteins, suggesting that these two proteins may be the products of two non-allelic XCK1(8) genes. The antibody does not cross-react with vimentin. We have used CK7 to follow the distribution of XCK1(8) throughout development by immunoblotting and immunocytochemistry. In larval stages, strong staining is seen in the notocord, the apical epithelia of the gut, the mesentery, and a few cells in the spinal cord. In oocytes and early embryos, two distinct intermediate filament (IF) networks can be distinguished: a cortical cytokeratin network, and a deeper vimentin one. In addition, the oocyte germ plasm stains with Z9 but not CK7. We propose that such distinct distributions of each IF protein reflect functional differences during early development.

Identification of vimentin and novel vimentin-related proteins in Xenopus oocytes and early embryos

Development ◽  
1990 ◽  
Vol 110 (4) ◽  
pp. 1185-1195
Author(s):  
N.P. Torpey ◽  
J. Heasman ◽  
C.C. Wylie
Keyword(s):  
Fusion Proteins ◽  
Xenopus Oocytes ◽  
Germ Plasm ◽  
Radial Glial Cells ◽  
Larval Stages ◽  
Early Embryos ◽  
Radial Glial ◽  
Synthetic Mrna ◽  
Related Proteins ◽  
First Time

We have made antibodies against fusion proteins of Xenopus vimentin. We show for the first time the distribution of vimentin in larval stages, where it is found in cells of mesenchymal origin, and in radial glial cells. In sections of Xenopus oocytes and early embryos, immunocytochemistry reveals the presence of an extensive cytoplasmic network, distributed in an animal-vegetal gradient. Germ plasm stains particularly strongly. The form of the IF proteins in this network is unusual. In immunoblot experiments the anti-vimentin antibodies detect a number of distinct proteins. We have identified those that are the products of the two known vimentin genes, by injection of synthetic mRNA transcribed from cloned vimentin cDNAs into oocytes, followed by two-dimensional Western blotting. This has demonstrated unambiguously that one Xenopus vimentin, Vim1, is present in oocytes and early embryos. However, two other immunoreactive proteins detected in Triton extracts of oocytes and early embryos are not the products of Vim1, since depletion of vimentin mRNA by antisense oligonucleotide injection has no effect on the synthesis of these proteins. These results suggest that novel IF-like proteins are expressed in Xenopus oocytes and early embryos.

Molecular cloning and characterization of a murine hemicholinium-3-sensitive choline transporter

2001 ◽  
Vol 29 (6) ◽  
pp. 711-716 ◽  
Author(s):  
S. Apparsundaram ◽  
S. M. Ferguson ◽  
R. D. Blakely
Keyword(s):  
Spinal Cord ◽  
Amino Acid ◽  
Genomic Dna ◽  
Cholinergic Neurons ◽  
Membrane Vesicles ◽  
Predicted Amino Acid Sequence ◽  
Sequence Information ◽  
Choline Uptake ◽  
Choline Transporter ◽  
Mouse Tissues

In cholinergic neurons, a specific requirement for precursor choline in the biosynthesis of acetylcholine (ACh) is thought to be sustained by a presynaptic, hemicholinium-3 (HC-3)-sensitive choline transporter (CHT). This transporter exhibits micromolar affinity for choline and transport activity is Na+- and Cl−-dependent. Based on the sequence information available with the recent cloning of rat and human CHTs [Okuda, Haga, Kanai, Endou, Ishihara and Katsura (2000) Nat. Neurosci. 3, 120–125; Apparsundaram, Ferguson, George Jr and Blakely (2000) Biochem. Biophys. Res. Commun. 276, 862–867; Okuda and Haga (2000) FEBS Lett. 484, 92–97], we have identified a murine CHT orthologue (mCHT) by reverse transcriptase-PCR of spinal cord mRNA and confirmed this sequence using assembled mouse genomic DNA. Inferred splice junctions for mCHT exons are conserved with those of hCHT. The mCHT cDNA encodes a protein of 580 amino acids with 93 % and 98 % amino acid identity to human CHT and rat CHT1, respectively. Hydropathy analysis of the predicted amino acid sequence of mCHT indicates a protein containing 13 transmembrane domains (TMDs), with the N-terminus oriented extracellularly and the C-terminus oriented intracellularly. Northern blot analysis of mouse tissues reveals the expression of mCHT as a single transcript of ~ 5 kb with highest expression in regions that are rich in cholinergic cell bodies, e.g. the spinal cord, brainstem, mid-brain and striatum, whereas hybridization signals are absent in regions lacking cholinergic soma, e.g. the cerebellum and kidney. Expression of mCHT in COS-7 cells results in high-affinity [3H]HC-3-binding sites (Kd = 5 nM), and Na+- and Cl−-dependent HC-3-sensitive choline uptake (Km = 2 μM), assessed in resealed membrane vesicles. The availability of cloned, functional mCHT and its cognate genomic DNA should prove useful for studies of mCHT regulation and should open possibilities for evaluation of CHT dysfunction in murine models.

Isolation and embryonic expression of an abdominal-A-like gene from the lepidopteran, Manduca sexta

Development ◽  
1991 ◽  
Vol 112 (1) ◽  
pp. 119-129 ◽  
Author(s):  
L.M. Nagy ◽  
R. Booker ◽  
L.M. Riddiford
Keyword(s):  
Amino Acid ◽  
Manduca Sexta ◽  
Abdominal Segment ◽  
Early Embryo ◽  
Predicted Amino Acid Sequence ◽  
Posterior Half ◽  
Larval Stages ◽  
Amino Acid Region ◽  
In Situ Hybridizations

Using sequence homology to the Drosophila Antennapedia gene, we isolated a homeobox-containing gene from the lepidopteran, Manduca sexta. Sequence analysis and in situ hybridizations to tissue sections suggest that the Manduca gene encodes a lepidopteran homologue of the Drosophila Bithorax complex gene abdominal-A. The predicted amino acid sequence of a 76 amino acid region that includes the homeobox and the regions immediately flanking it are identical between the Manduca and Drosophila genes. Northern blots reveal that the manduca abd-A gene is expressed first in the early embryo and continues to be expressed throughout later embryonic and larval stages. In situ hybridizations show that the posterior half of the first abdominal segment marks the anterior border of the Manduca abd-A expression. This expression pattern demonstrates the conservation of parasegments as domains of gene activity in the lepidopteran embryo. The Manduca abd-A expression extends from the posterior half of the first abdominal segment through the tenth abdominal segment, a domain that is greater than that of the Drosophila abd-A expression, and reflects the difference in visible segment number between the two insects.

scholarly journals Commissural Interneurons in Rhythm Generation and Intersegmental Coupling in the Lamprey Spinal Cord

1999 ◽  
Vol 81 (5) ◽  
pp. 2037-2045 ◽  
Author(s):  
James T. Buchanan
Keyword(s):  
Spinal Cord ◽  
Amino Acid ◽  
Excitatory Amino Acid ◽  
Rhythmic Activity ◽  
Ventral Root ◽  
Rhythm Generation ◽  
Spinal Segment ◽  
Spinal Segments ◽  
Autocorrelation Method

Commissural interneurons in rhythm generation and intersegmental coupling in the lamprey spinal cord. To test the necessity of spinal commissural interneurons in the generation of the swim rhythm in lamprey, longitudinal midline cuts of the isolated spinal cord preparation were made. Fictive swimming was then induced by bath perfusion with an excitatory amino acid while recording ventral root activity. When the spinal cord preparation was cut completely along the midline into two lateral hemicords, the rhythmic activity of fictive swimming was lost, usually replaced with continuous ventral root spiking. The loss of the fictive swim rhythm was not due to nonspecific damage produced by the cut because rhythmic activity was present in split regions of spinal cord when the split region was still attached to intact cord. The quality of this persistent rhythmic activity, quantified with an autocorrelation method, declined with the distance of the split spinal segment from the remaining intact spinal cord. The deterioration of the rhythm was characterized by a lengthening of burst durations and a shortening of the interburst silent phases. This pattern of deterioration suggests a loss of rhythmic inhibitory inputs. The same pattern of rhythm deterioration was seen in preparations with the rostral end of the spinal cord cut compared with those with the caudal end cut. The results of this study indicate that commissural interneurons are necessary for the generation of the swimming rhythm in the lamprey spinal cord, and the characteristic loss of the silent interburst phases of the swimming rhythm is consistent with a loss of inhibitory commissural interneurons. The results also suggest that both descending and ascending commissural interneurons are important in the generation of the swimming rhythm. The swim rhythm that persists in the split cord while still attached to an intact portion of spinal cord is thus imposed by interneurons projecting from the intact region of cord into the split region. These projections are functionally short because rhythmic activity was lost within approximately five spinal segments from the intact region of spinal cord.

Estradiol inhibits the activity of proton-coupled amino acid transporter PAT1 expressed in Xenopus oocytes

2012 ◽  
Vol 695 (1-3) ◽  
pp. 34-39
Author(s):  
Linlin Shan ◽  
Yujie Yang ◽  
Jin Wang ◽  
Ji Zuo ◽  
Xianhong Dong ◽  
...  
Keyword(s):  
Amino Acid ◽  
Xenopus Oocytes ◽  
Amino Acid Transporter ◽  
Acid Transporter

scholarly journals Multiple components of arginine and phenylalanine transport induced in neutral and basic amino acid transporter-cRNA-injected Xenopus oocytes

1996 ◽  
Vol 318 (3) ◽  
pp. 915-922 ◽  
Author(s):  
George J PETER ◽  
Iain G. DAVIDSON ◽  
Aamir AHMED ◽  
Lynn McILROY ◽  
Alexander R. FORRESTER ◽  
...  
Keyword(s):  
Amino Acid ◽  
Xenopus Oocytes ◽  
Competitive Inhibition ◽  
Protein Complexes ◽  
Basic Amino Acid ◽  
Amino Acid Transporter ◽  
Transport Processes ◽  
Arginine Transport ◽  
Acid Transporter ◽  
Phenylalanine Transport

The induced uptakes of l-[3H]phenylalanine and l-[3H]arginine in oocytes injected with clonal NBAT (neutral and basic amino acid transporter) cRNA show differential inactivation by pre-treatment with N-ethylmaleimide (NEM), revealing at least two distinct transport processes. NEM-resistant arginine transport is inhibited by leucine and phenylalanine but not by alanine or valine; mutual competitive inhibition of NEM-resistant uptake of arginine and phenylalanine indicates that the two amino acids share a single transporter. NEM-senstive arginine transport is inhibited by leucine, phenylalanine, alanine and valine. At least two NEM-sensitive transporters may be expressed because we have been unable to confirm mutual competitive inhibition between arginine and phenylalanine transport. The NEM-resistant transport mechanism appears to involve distinct but overlapping binding sites for cationic and zwitterionic substrates. NBAT is known to form oligomeric protein complexes in cell membranes, and its functional roles when expressed in Xenopus oocytes may include interaction with oocyte proteins, leading to increased native amino acid transport activities; these resemble NBAT-expressed activities in terms of NEM-sensitivity and apparent substrate range (including an unusual inhibition by β-phenylalanine).

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